Rsv f protein compositions and methods for making same

ABSTRACT

The present invention relates to immunogenic compositions comprising RSV F protein, methods for preparing compositions that contain RSV F protein ecto-domain polypeptides, and to certain engineered RSV F proteins and nucleic acids that encode the engineered RSV F proteins. Compositions prepared using the methods can contain RSV F protein ecto-domain polypeptides in a predominant or single desired form and conformation. The invention also relates to methods for inducing an immune response to RSV F.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. patent application Ser. No.17/564,962, filed on Dec. 29, 2021, which is a Continuation of U.S.patent application Ser. No. 15/678,798, filed on Aug. 16, 2017 (now U.S.Pat. No. 11,261,239, issued on Mar. 1, 2022), which is a Continuation ofU.S. patent application Ser. No. 12/836,931, filed on Jul. 15, 2010,which claims the benefit of U.S. Patent Application No. 61/225,805,filed on Jul. 15, 2009, and U.S. Patent Application No. 61/294,426,filed on Jan. 12, 2010. The entire teachings of the above applicationsare incorporated herein by reference.

BACKGROUND OF THE INVENTION

Respiratory syncytial virus (RSV) is an enveloped non-segmentednegative-strand RNA virus in the family Paramyxoviridae, genusPneumovirus. It is the most common cause of bronchiolitis and pneumoniaamong children in their first year of life. RSV also causes repeatedinfections including severe lower respiratory tract disease, which mayoccur at any age, especially among the elderly or those with compromisedcardiac, pulmonary, or immune systems.

To infect a host cell, paramyxoviruses such as RSV, like other envelopedviruses such as influenza virus and HIV, require fusion of the viralmembrane with a host cell's membrane. For RSV the conserved fusionprotein (RSV F) fuses the viral and cellular membranes by couplingirreversible protein refolding with juxtaposition of the membranes. Incurrent models based on paramyxovirus studies, the RSV F proteininitially folds into a metastable “pre-fusion” conformation. During cellentry, the pre-fusion conformation undergoes refolding andconformational changes to its stable “post-fusion” conformation.

The RSV F protein is translated from mRNA into an approximately 574amino acid protein designated F₀. Post-translational processing of F₀includes removal of an N-terminal signal peptide by a signal peptidasein the endoplasmic reticulum. F₀ is also cleaved at two sites(approximately 109/110 and approximately 136/137) by cellular proteases(in particular furin) in the trans-Golgi. This cleavage results in theremoval of a short intervening sequence and generates two subunitsdesignated F₁ (˜50 kDa; C-terminal; approximately residues 137-574) andF₂ (˜20 kDa; N-terminal; approximately residues 1-109) that remainassociated with each other. F₁ contains a hydrophobic fusion peptide atits N-terminus and also two amphipathic heptad-repeat regions (HRA andHRB). HRA is near the fusion peptide and HRB is near the transmembranedomain. Three F₁-F₂ heterodimers are assembled as homotrimers of F₁-F₂in the virion.

A vaccine against RSV infection is not currently available, but isdesired. One potential approach to producing a vaccine is a subunitvaccine based on purified RSV F protein. However, for this approach itis desirable that the purified RSV F protein is in a single form andconformation that is stable over time, consistent between vaccine lots,and conveniently purified.

The RSV F protein can be truncated, for example by deletion of thetransmembrane domain and cytoplasmic tail, to permit its expression asan ectodomain, which may be soluble. In addition, although RSV F proteinis initially translated as a monomer, the monomers are cleaved andassemble into trimers. When RSV F protein is in the form of cleavedtrimers, the hydrophobic fusion peptide is exposed. The exposedhydrophobic fusion peptides on different trimers, e.g., solubleecto-domain trimers, can associate with each other, resulting in theformation of rosettes. The hydrophobic fusion peptides can alsoassociate with lipids and lipoproteins, for example from cells that areused to express recombinant soluble RSV F protein. Due to the complexityof RSV F protein processing, structure and refolding, purified,homogeneous, immunogenic preparations are difficult to obtain.

Thus, there is a need for improved RSV F protein compositions andmethods for making RSV F protein compositions.

SUMMARY OF THE INVENTION

The invention relates to immunogenic compositions that contain one ormore RSV F polypeptides, and to certain engineered RSV F proteins andnucleic acids that encode the engineered RSV F proteins.

In one aspect the RSV F protein is soluble. For example, the RSV Fprotein can have the transmembrane region and cytoplasmic tail deleted.In some aspects, the soluble RSV F contains one or more of 1) one ormore mutations to one or both furin-cleavage sites, 2) one or moremutations to the fusion peptide, 3) one or more mutations to the p27linker, 4) contains an added oligomerization sequence, and 5) containsan added amino acid sequence that provides a protease cleavage site. Inadditional or alternative aspects, the RSV F protein is a monomer, atrimer, or a combination of monomers and trimers. The trimer can bemonodispered or in the form of a rosette. In further additional oralternative aspects, the RSV F protein can be in a prefusionconformation, an intermediate conformation or a postfusion conformation.

In one aspect, the immunogenic composition contains one or morerespiratory syncytial virus F (RSV F) polypeptides in which amino acids100-150 are replaced with the amino acid sequence of SEQ ID NO:9, SEQ IDNO:12, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7;SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:91 orSEQ ID NO:92. In some embodiments the RSV F polypeptide is soluble(e.g., an ectodomain).

In another aspect, the immunogenic composition contains an RSV Fpolypeptide in which amino acids 100-150 of the RSV F are replaced withthe amino acid sequence of SEQ ID NO:12. In some embodiments the RSV Fpolypeptide is soluble (e.g., an ectodomain).

In yet another aspect, the immunogenic composition contains an RSV Fpolypeptide in which amino acids 100-150 of the RSV F are replaced withthe amino acid sequence of SEQ ID NO:9, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7; SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11,SEQ ID NO:13, or SEQ ID NO:92. In some embodiments the RSV F polypeptideis soluble (e.g., an ectodomain).

In another aspect, the immunogenic composition contains an RSV Fpolypeptide in which amino acids 100-150 are replaced with the aminoacid sequence of SEQ ID NO:9. In some embodiments the RSV F polypeptideis soluble (e.g., an ectodomain).

In another aspect, the immunogenic composition contains an RSV Fpolypeptide in which RSV F contains amino acids 23-99 and 151-524 of SEQID NO:1 or SEQ ID NO:2. In some embodiments the RSV F polypeptide issoluble (e.g., an ectodomain).

In one aspect, the immunogenic composition contains a polypeptideselected from the group consisting of SEQ ID NO:49, SEQ ID NO:68, SEQ IDNO:71, SEQ ID NO: 25, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ IDNO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:41, SEQ IDNO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ IDNO:47, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ IDNO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:58, SEQ IDNO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ IDNO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:69, SEQ IDNO:70, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ IDNO:89, and SEQ ID NO:93. In some embodiments, the signal peptide and/orHIS tag is omitted. In some embodiments the RSV F polypeptide is soluble(e.g., an ectodomain).

In one aspect, the immunogenic composition contains SEQ ID NO:68 oralternatively, SEQ ID NO:68 in which the signal peptide, and optionallythe HIS tag, is omitted.

In another aspect, the immunogenic composition contains a polypeptideselected from the group consisting of SEQ ID NO:49, SEQ ID NO:71, andany of the foregoing sequences in which the signal peptide, andoptionally the HIS tag, is omitted. In some embodiments the RSV Fpolypeptide is soluble (e.g., an ectodomain).

In preferred embodiments, the immunogenic composition will include anadjuvant. The adjuvant is preferably an aluminum salt, asqualene-in-water emulsion (such as MF59), a benzonaphthyridinecompound, a phospholipid compound (such as E6020), a small moleculeimmunepotentiator or a combination of any two or more of any of theforegoing.

Yet another aspect of the invention includes recombinant RSV Fpolypeptides. The RSV F may be in the form of a monomer, trimer, rosetteof trimers, or combination of monomers and trimers. The recombinantpolypeptide may include a heterologous oligomerization domain, anepitope or a signal peptide. The heterologous oligomerization domain ispreferably a trimerization domain from influenza hemagglutinin, fromSARS spike, or from HIV gp41, NadA, modified GCN4, or ATCase.

In one aspect, the recombinant RSV F polypeptide has amino acids 100-150replaced with the amino acid sequence of SEQ ID NO:9, SEQ ID NO:12, SEQID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ IDNO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:91 or SEQ IDNO:92. In some embodiments the RSV F polypeptide is soluble (e.g., anectodomain).

In another aspect, the recombinant RSV F polypeptide has amino acids100-150 of the RSV F replaced with the amino acid sequence of SEQ IDNO:12. In some embodiments the RSV F polypeptide is soluble (e.g., anectodomain).

In another aspect, the recombinant RSV F polypeptide has amino acids100-150 of the RSV F replaced with the amino acid sequence of SEQ IDNO:9, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7;SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:13, or SEQ ID NO:92.In some embodiments the RSV F polypeptide is soluble (e.g., anectodomain).

In yet another aspect, the recombinant RSV F polypeptide has amino acids100-150 of the RSV F replaced with the amino acid sequence of SEQ IDNO:9. In some embodiments the RSV F polypeptide is soluble (e.g., anectodomain).

In one aspect, the recombinant polypeptide is selected from the groupconsisting of SEQ ID NO:49, SEQ ID NO:68, SEQ ID NO:71, SEQ ID NO: 25,SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33,SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43,SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:47,SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54,SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60,SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65,SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:85,SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:93,and any combinations thereof. Optionally, the signal peptide and/or HIStag is omitted. In some embodiments the RSV F polypeptide is soluble(e.g., an ectodomain).

Still another aspect includes nucleic acids encoding any of theforegoing polypeptides. The nucleic acid may be a self-replicating RNAmolecule.

Another aspect of the invention is an immunogenic composition comprisinga self-replicating RNA that encodes an RSV F polypeptide. Theimmunogenic composition can include a delivery system.

Another aspect of the invention includes methods of inducing an immuneresponse to RSV F by administering any of the immunogenic compositions.

The invention relates to methods for preparing compositions and tocompositions that contain RSV F protein, such as soluble RSV Fecto-domain polypeptides, including immunogenic compositions. The RSV Fecto-domain polypeptides can be in a single form, such as uncleavedmonomers, uncleaved trimers, cleaved trimers, or rosettes of cleavedtrimers. The RSV F ectodomain polypeptides can also be in two or moreforms, for example two or more forms that exist in equilibrium, such asequilibrium between uncleaved monomers and uncleaved trimers. Theinvention provides several advantages. For example, the presence of asingle desired form of RSV F in an immunogenic composition provides amore predictable immune response when the composition is administered toa subject, and more consistent stability and other physical and chemicalcharacteristics when formulated into a vaccine.

In one aspect, the invention is a method for producing a compositioncomprising cleaved RSV F protein ecto-domain polypeptides. The methodincludes a) providing uncleaved RSV F protein ecto-domain polypeptidescontaining one or more protease cleavage sites that, when cleaved,produce F₁ and F₂ fragments, and b) cleaving the uncleaved RSV F proteinecto-domain polypeptides with a protease or proteases that recognize theprotease cleavage site or sites. In general, the amino acid sequence ofthe uncleaved RSV F protein ecto-domain polypeptides contains alteredfurin cleavage sites, and the RSV F protein ecto-domain polypeptides aresecreted from a host cell that produces them uncleaved at a positionfrom amino acid 101 to amino acid 161, (e.g., is not cleaved at thefurin cleavage sites at positions 106-109 and 131-136). In someembodiments, the uncleaved RSV F protein ecto-domain polypeptidesprovided in a) are purified.

The uncleaved RSV F protein ecto-domain polypeptides provided in a) cancomprise an intact fusion peptide or an altered fusion peptide (e.g., adeleted fusion peptide or mutated fusion peptide). When the uncleavedRSV F protein ecto-domain polypeptides provided in a) contain an intactfusion peptide, the cleaving in step b) results in the formation ofrosettes of trimers. When the uncleaved RSV F protein ecto-domainpolypeptides provided in a) comprise an altered fusion peptide, thecleaving in step b) results in the formation of trimers.

The method can further comprise the optional step of purifying therosettes or trimers produced by cleaving the uncleaved RSV F proteinecto-domain polypeptides. In preferred embodiments, the cleaved RSV Fprotein ecto-domain polypeptides produced according to the method aresubstantially free of lipids and lipoproteins.

In another aspect, the invention is a method for producing a compositioncomprising uncleaved RSV F protein ecto-domain polypeptide monomers,trimers or a combination of monomers and trimers. The method includes a)providing a biological material that contains uncleaved RSV F proteinecto-domain polypeptides; and b) purifying uncleaved RSV F proteinecto-domain polypeptide monomers or trimers from the biologicalmaterial. In general, the amino acid sequence of the uncleaved RSV Fprotein ecto-domain polypeptides contains altered furin cleavage sites,and the RSV F protein ecto-domain polypeptides are secreted from a hostcell that produces them uncleaved at a position from amino acid 101 toamino acid 161, (e.g., is not cleaved at the furin cleavage sites atpositions 106-109 and 131-136). In some embodiments, the amino acidsequence of the uncleaved RSV F protein ecto-domain polypeptides furthercontain altered trypsin cleavage sites, and the RSV F proteinecto-domain polypeptides are not cleaved by trypsin at a site betweenamino acid 101 and amino acid 161. In other embodiments, the amino acidsequence of the uncleaved RSV F protein ecto-domain polypeptides furthercontain an altered fusion peptide.

In some embodiments, uncleaved RSV F protein ecto-domain polypeptidetrimers are purified. In other embodiments, uncleaved RSV F proteinecto-domain polypeptide monomers are purified. In still otherembodiments a mixture of uncleaved RSV F protein ecto-domain monomersand trimers, which may be in a dynamic equilibrium, are purified. Inpreferred embodiments, the cleaved RSV F protein ecto-domainpolypeptides produced according to the method are substantially free oflipids and lipoproteins.

In another aspect, the invention is a method for producing a compositioncomprising cleaved RSV F protein ecto-domain polypeptide monomers,trimers or a combination of monomers and trimers. The method includes a)providing a biological material that contains cleaved RSV F proteinecto-domain polypeptides that contain an altered fusion peptide; and b)purifying cleaved RSV F protein ecto-domain polypeptides from thebiological material.

In some embodiments, cleaved RSV F protein ecto-domain polypeptidetrimers are purified. In other embodiments, cleaved RSV F proteinecto-domain polypeptide monomers are purified. In still otherembodiments a mixture of cleaved RSV F protein ecto-domain monomers andtrimers, which may be in a dynamic equilibrium, are purified. Inpreferred embodiments, the cleaved RSV F protein ecto-domainpolypeptides produced according to the method are preferablysubstantially free of lipids and lipoproteins. In still anotherembodiment, a cleaved RSV F protein ectodomain trimer containing analtered fusion peptide is purified.

In other aspects, the invention provides compositions, includingimmunogenic compositions, produced using the methods of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C shows the schematic of the wild type RSV F (FIG. 1A) and ofa ectodomain construct in which the transmembrane domain and cytoplasmictail have been removed and an optional HIS6-tag has been added to theC-terminus (FIG. 1B). For clarity, residue numbering is related to thewild type A2 strain RSV F beginning at the N-terminal signal peptide andis not altered in constructs containing amino acid deletions. Labeled inthe schematics is the signal sequence or signal peptide (sp). FIG. 1A isa schematic of RSV F protein showing the signal sequence or signalpeptide (SP), p27 linker region, fusion peptide (FP), HRA domain (HRA),HRB domain (HRB), transmembrane region (TM), and cytoplasmic tail (CT).The C-terminal bounds of the ectodomain can very. FIG. 1B is a generalschematic of the RSV F ectodomain construct depicting the sharedfeatures with the schematics in FIG. 1A and including an optionalHIS6-tag (HIS TAG). Furin cleavage sites are present at amino acidpositions 109/110 and 136/137. FIG. 1C also shows the amino acidsequence of amino acids 100-150 of RSV F (wild type) (SEQ ID NO:108) andseveral proteins (Furmt—SEQ ID NO:3; Furdel—SEQ ID NO:4; Furx—SEQ IDNO:6; Furx R113Q, K123N, K124N—SEQ ID NO:5; Furx R113Q, K123Q, K124Q—SEQID NO:92; Delp21 furx—SEQ ID NO:7; Delp23 furx—SEQ ID NO: 8; Delp23furdel—SEQ ID NO:9; N-Term Furin—SEQ ID NO:10; C-term Furin—SEQ ID NO:11; Fusion Peptide Deletion1—SEQ ID NO:12; and Factor Xa—SEQ ID NO:13)in which the one or both furin cleavage sites and/or fusion peptideregion were mutated or deleted. In FIG. 1C, the symbol “-” indicatesthat the amino acid at that position is deleted.

FIG. 2 shows the amino acid sequence of the carboxy terminus from aminoacid position 488 to the start of the TM region of RSV F (wild type)(SEQ ID NO:94) and several proteins (SEQ ID NOS:95-100) that containadded protease cleavage sites. In FIG. 2, the symbol “-” indicates thatthere is no amino acid at that position.

FIG. 3 is a chromatogram and image of an electrophoresis gel showing thepurification of RSV F monomers (3) using size exclusion chromatography.

FIGS. 4A-4F shows the nucleotide sequence (SEQ ID NO:101) of the plasmidencoding the pT7-TC83R-FL.RSVF (A317) self-replicating RNA moleculewhich encodes the respiratory syncytial virus F glycoprotein (RSV-F).The nucleotide sequence encoding RSV-F is underlined.

FIG. 5 is an alignment of the amino acid sequences of F proteins fromseveral strains of RSV. The alignment was prepared using the algorithmdisclosed by Corpet, Nucleic Acids Research, 1998, 16(22):10881-10890,using default parameters (Blossum 62 symbol comparison table, gap openpenalty: 12, gap extension penalty: A2, F protein of the strain A2(accession number AF035006) (SEQ ID NO:102); CP52, F protein of the CP52strain (accession number AF013255) (SEQ ID NO:103); B, F protein of theB strain (accession number AF013254) (SEQ ID NO:104); long, F protein ofthe long strain (accession number AY911262) strain (SEQ ID NO:105), and18537 strain, F protein of the 18537 strain (accession number Swiss ProtP13843) (SEQ ID NO:106). A consensus of F protein sequences is alsoshown (SEQ ID NO:107)

FIGS. 6A-6D shows relevant regions of size exclusion (SEC) chromatogramsfrom select RSV F antigen purifications. The principle peak containingthe indicated antigen is indicated by an asterisk with the retentiontime of the Superdex P200 16/60 column (GE Healthcare) is indicated inmilliliters. On a calibrated column, the approximate retention times of47 mls, 65 mls and 77 mls correspond to the column void volume, theretention of the RSV F trimer and the retention of the RSV F monomer,respectively. In FIG. 6A, the uncleaved Delp23 Furdel (Δp23 Furdel)construct is purified from the monomer peak at approximately 77 mls.When the uncleaved Delp23 Furdel RSV F antigen is treated with trypsin,the protein can form rosettes, which now migrate on SEC in the voidvolume at approximately 47 mls (FIG. 6B). The cleaved trimer species ofRSV F fusion peptide deletion is purified from the trimer peak atapproximately 65 mls retention time (FIG. 6C) while the uncleaved Delp21Furx construct (Δp21 Furx) is purified from the monomer peak atapproximately 77 mls (FIG. 6D).

FIGS. 7A-7D shows representative EM images of select RSV F antigens.FIG. 7A shows an EM image of RSV F Δp23 (Delp23) before trypsintreatment. The crutch shapes in FIG. 7A, consistent with a postfusiontrimer conformation, are not always observed in the uncleaved Δp23(Delp23) Furdel construct. When the Δp23 (Delp23) Furdel construct istreated with trypsin and purified from the void volume of an SEC columnand observed by EM the proteins are found to have adopted rosetteconformations (FIG. 7B). When the RSV F fusion peptide deletionconstruct is purified from the trimer peak on an SEC column amonodispersed crutch shape is observed, consistent with the a postfusiontrimer (FIG. 7C). Shown in FIG. 7D are three preparations of either Δp21(Delp21) furx RSV F (labeled Monomer), Fusion peptided deletion RSV F(lanes labeled Trimer) and purified RSV F rosettes (labeled Rosettes).The gel contains several lanes of GE Full Range Standard (molecularweights standard are labeled to the left of the gel) while approximateretention times of RSV F fragments are indicated on the right of thegel.

FIGS. 8A-8C are graphs showing that monomers (uncleaved Δp21 (Delp21)furx), rosettes of trimers (cleaved Δp23 (Delp23) Furdel), and trimers(fusion peptide deletion) of RSV F ecto-domain polypeptides areimmunogenic in cotton rats. Serum titers of anti-RSV F IgG andneutralizing anti-RSV antibodies were measured 2 weeks after the 1^(st)vaccination (2wp1), 3 weeks after the 1^(st) vaccination (3wp1), and/or2 weeks after the 2^(nd) vaccination (2wp2).

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to respiratory syncytial virus F (RSV F)polypeptides and/or proteins, immunogenic compositions comprising RSV Fpolypeptides and/or proteins, methods for producing RSV F polypeptidesand/or proteins and compositions comprising RSV F polypeptides and/orproteins, and nucleic acids that encode RSV F polypeptides and/orproteins.

In general, the immunogenic compositions comprise RSV F polypeptidesand/or proteins that contain mutations (e.g., amino acid replacements,deletions or additions) which provide beneficial characteristics, suchas one or more of 1) stabilized prefusion or intermediate (non-postfusion) conformation, 2) reduced or eliminated exposure of the fusionpeptide, 3) improved stability (e.g., reduced aggregation and/ordegradation, and 4) more closely resemble active F₁/F₂ viral protein.These characteristics provide advantages for the immunogeniccompositions and for the manufacture of the immunogenic compositions.For example, as described herein, non-post fusion conformations of RSV Fprotein (i.e., prefusion conformation, intermediate conformations) canbe better immunogens and elicit a better neutralizing antibody response.Reducing or eliminating the exposure of the fusion peptide, e.g., byintroducing mutations or deletions into the furin cleavage sites, willreduce the hydrophobicity of the polypeptide and facilitatepurifications, and also reduce or eliminate the RSV F protein fromassociating with cell membranes in a subject to whom the protein isadministered. Improved stability of the protein facilitates producingimmunogenic compositions in which the protein has a decreased tendencyto aggregate or degrade, which provides a more predictable immuneresponse when the composition is administered to a subject. Finally,mutant RSV F polypeptides or proteins that resemble F1/F2 viral protein,for example by deletion of all or part of the p27 linker region, mayelicit a better neutralizing antibody response. Other advantages of theinvention are described herein.

The invention also relates to methods for preparing compositions thatcontain RSV F protein, in particular RSV F ecto-domain polypeptides, andto compositions including immunogenic compositions comprising RSV Fprotein. Preferably, the RSV F ecto-domain polypeptides are in a singleform or in a dynamic equilibrium between known forms.

Definitions

As used herein “population” refers to more than one RSV F polypeptide orprotein that is present in a composition. The population can besubstantially homogenous, in which substantially all RSV F polypeptidesor proteins are substantially the same (e.g., same amino acid sequence,same conformation), heterogenous, or have a desired degree ofhomogenicity (e.g., at least about 50%, at least about 55%, at leastabout 60%, at least about 65%, at least about 70%, at least about 75%,at least about 80%, at least about 85%, at least about 90%, at leastabout 95%, at least about 99% of the RSV F polypeptides or proteins areprefusion conformation, are postfusion conformation, are monomers, aretrimers).

The “post fusion conformation” of RSV F protein is a trimercharacterized by the presence of a six-helix bundle comprising 3 HRB and3HRA regions.

The “pre-fusion conformation” of RSV F protein is a conformationcharacterized by a trimer that contains a triple helix comprising 3 HRBregions.

As used herein, “RSV F ecto-domain polypeptide” refers to an RSV Fprotein polypeptide that contains substantially the extracellularportion of mature RSV F protein, with our without the signal peptide(e.g., about amino acids 1 to about amino acid 524, or about amino acid22 to about amino acid 524) but lacks the transmembrane domain andcytoplasmic tail of naturally occurring RSV F protein.

As used herein, “cleaved RSV F ecto-domain polypeptide” refers to a RSVF ectodomain polypeptide that has been cleaved at one or more positionsfrom about 101/102 to about 160/161 to produce two subunits, in whichone of the subunits comprises F₁ and the other subunit comprises F₂.

As used herein, “C-terminal uncleaved RSV F ecto-domain polypeptide”refers to an RSV F ectodomain polypeptide that is cleaved at one or morepositions from about 101/102 to about 131/132, and is not cleaved at oneor more positions from about 132/133 to about 160/161, to produce twosubunits, in which one of the subunits comprises F₁ and the othersubunit comprises F₂.

As used herein, “uncleaved RSV F ecto-domain polypeptide” refers to anRSV F ectodomain polypeptide that is not cleaved at one or morepositions from about 101/102 to about 160/161. An uncleaved RSV Fecto-domain polypeptide can be, for example, a monomer or a trimer.

As used herein, “fusion peptide” refers to amino acids 137-154 of RSV Fprotein.

As used herein, “altered fusion peptide” refers to a fusion peptide inwhich one or more amino acids are independently replaced or deleted,including replacement or deletion of all amino acids from positions137-154. Preferably, cleaved RSV F ecto-domain polypeptides that containan “altered fusion peptide” do not form rosettes.

As used herein, a “purified” protein or polypeptide is a protein orpolypeptide which is recombinantly or synthetically produced, orproduced by its natural host, and has been isolated from othercomponents of the recombinant or synthetic production system or naturalhost such that the amount of the protein relative to othermacromolecular components present in a composition is substantiallyhigher than that present in a crude preparation. In general, a purifiedprotein will be at least about 50% homogeneous and more preferably atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95% or substantially homogeneous.

As used herein, “substantially free of lipids and lipoproteins” refersto compositions, proteins and polypeptides that are at least about 95%free of lipids and lipoproteins on a mass basis when protein and/orpolypeptide (e.g., RSV F polypeptide) purity is observed on an SDS PAGEgel and total protein content is measured using either UV280 absorptionor BCA analysis, and lipid and lipoprotein content is determined usingthe Phospholipase C assay (Wako, code no. 433-36201).

As used herein, “altered furin cleavage site” refers the amino acidsequence at about positions 106-109 and at about positions 133-136 innaturally occurring RSV F protein that are recognized and cleaved byfurin or furin-like proteases, but in an uncleaved RSV F proteinecto-domain polypeptide contains one or more amino acid replacements,one or more amino acid deletions, or a combination of one or more aminoacid replacement and one or more amino acid deletion, so that an RSV Fecto-domain polypeptide that contains an altered furin cleavage site issecreted from a cell that produces it uncleaved at the altered furincleavage site.

Features of RSV F protein ecto-domains suitable for use in thisinvention are described herein with reference to particular amino acidsthat are identified by the position of the amino acid in the sequence ofRSV F protein from the A2 strain (SEQ ID NO:1). RSV F proteinecto-domains can have the amino acid sequence of the F protein from theA2 strain or any other desired strain. When the F protein ecto-domainfrom a strain other than the A2 strain is used, the amino acids of the Fprotein are to be numbered with reference to the numbering of the Fprotein from the A2 strain, with the insertion of gaps as needed. Thiscan be achieved by aligning the sequence of any desired RSV F proteinwith the F protein of the strain A2, as shown herein for F proteins fromthe A2 strain, CP52 strain, B strain, long strain, and the 18537 strain.See, FIG. 5. Sequence alignments are preferably produced using thealgorithm disclosed by Corpet, Nucleic Acids Research, 1998,16(22):10881-10890, using default parameters (Blossum 62 symbolcomparison table, gap open penalty: 12, gap extension penalty: 2).

The invention provides soluble RSV F polypeptides and proteins, andimmunogenic compositions comprising the soluble RSV F polypeptides andproteins, as well as compositions comprising nucleic acids (e.g.,self-replicating RNA molecules) that encode the soluble RSV Fpolypeptides and proteins.

The RSV F polypeptides (e.g., ecto-domain polypeptides) can be in anydesired form, such as in a single form, such as uncleaved monomers,uncleaved trimers, cleaved trimers, or rosettes of cleaved trimers. TheRSV F ectodomain polypeptides can also be in two or more forms, forexample two or more forms that exist in equilibrium, such as equilibriumbetween uncleaved monomers and uncleaved trimers. The invention providesseveral advantages. For example, the presence of a single desired formof RSV, or a dynamic equilibrium between known forms, in an immunogeniccomposition, provides for more predictable formulation, solubility andstability, and for a more predictable immune response when thecomposition is administered to a subject.

Preferably, the RSV F ecto-domain polypeptides are in a single form,such as uncleaved monomers, uncleaved trimers, cleaved trimers, rosettesof cleaved trimers, or in a dynamic equilibrium between a subset of suchforms (e.g., equilibrium between uncleaved monomers and uncleavedtrimers).

In one aspect of the invention, the RSV F polypeptides and proteins arein pre-fusion conformation. The epitopes of the pre-fusion conformationmay be better able to elicit antibodies that can recognize andneutralize natural virions.

In one embodiment of the invention an immunogenic composition comprisesa population of respiratory syncytial virus F glycoproteins inpre-fusion conformation. In another aspect of the invention, animmunogenic composition comprises a population of respiratory syncytialvirus F glycoproteins which disfavor the post-fusion conformation ascompared to a population of isolated RSV F glycoproteins.

The invention also provides an immunogenic composition comprising apolypeptide that displays an epitope present in a pre-fusion or anintermediate fusion conformation of respiratory syncytial virus Fglycoprotein but absent the glycoprotein's post-fusion conformation.

The F Glycoprotein

The F glycoprotein of RSV directs viral penetration by fusion betweenthe virion envelope and the host cell plasma membrane. It is a type Isingle-pass integral membrane protein having four general domains:N-terminal ER-translocating signal sequence (SS), ectodomain (ED),transmembrane domain (TM), and a cytoplasmic tail (CT). CT contains asingle palmitoylated cysteine residue. The sequence of F protein ishighly conserved among RSV isolates, but is constantly evolving (7).Unlike most paramyxoviruses, the F protein in RSV can mediate entry andsyncytium formation independent of the other viral proteins (HN isusually necessary in addition to F in other paramyxoviruses).

The hRSV F mRNA is translated into a 574 amino acid precursor proteindesignated F₀, which contains a signal peptide sequence at theN-terminus that is removed by a signal peptidase in the endoplasmicreticulum. F₀ is cleaved at two sites (a.a. 109/110 and 136/137) bycellular proteases (in particular furin) in the trans-Golgi, removing ashort glycosylated intervening sequence and generating two subunitsdesignated F₁ (˜50 kDa; C-terminus; residues 137-574) and F₂ (˜20 kDa;N-terminus; residues 1-109) (See, e.g., FIGS. 1A-1C). F₁ contains ahydrophobic fusion peptide at its N-terminus and also two hydrophobicheptad-repeat regions (HRA and HRB). HRA is near the fusion peptide andHRB is near to the transmembrane domain (See, e.g., FIGS. 1A-1C). TheF₁-F₂ heterodimers are assembled as homotrimers in the virion.

RSV exists as a single serotype but has two antigenic subgroups: A andB. The F glycoproteins of the two groups are about 90% identical. The Asubgroup, the B subgroup, or a combination or hybrid of both can be usedin the invention. An example sequence for the A subgroup is SEQ ID NO: 1(A2 strain; GenBank GI: 138251; Swiss Prot P03420), and for the Bsubgroup is SEQ ID NO: 2 (18537 strain; GI: 138250; Swiss Prot P13843).SEQ ID NO:1 and SEQ ID NO:2 are both 574 amino acid sequences. Thesignal peptide in A2 strain is a.a. 1-21, but in 18537 strain it is1-22. In both sequences the TM domain is from about a.a. 530-550, buthas alternatively been reported as 525-548.

SEQ ID NO: 1   1MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIE  60  61LSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPPTNNRARRELPRFMNYTLN 120 121NAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVS 180 181LSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVN 240 241AGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYV 300 301VQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKV 360 361QSNRVFCDTMNSLTLPSEINLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKT 420 421KCTASNKNRGIIKTFSNGCDYVSNKGMDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDP 480 481LVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLS 540 541LIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN 574 SEQ ID NO: 2   1MELLIHRSSAIFLTLAVNALYLTSSQNITEEFYQSTCSAVSRGYFSALRTGWYTSVITIE  0  61LSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNRARREAPQYMNYTIN 120 121TTKNLNVSISKKRKRRFLGFLLGVGSAIASGIAVSKVLHLEGEVNKIKNALLSTNKAVVS 180 181LSNGVSVLTSKVLDLKNYINNRLLPIVNQQSCRISNIETVIEFQQMNSRLLEITREFSVN 240 241AGVTTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYV 300 301VQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKV 360 361QSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKT 420 421KCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDP 480 481LVFPSDEFDASISQVNEKINQSLAFIRRSDELLHNVNTGKSTTNIMITTIIIVIIVVLLS 540 541LIAIGLLLYCKAKNTPVTLSKDQLSGINNIAFSK 574

The invention may use any desired RSV F amino acid sequence, such as theamino acid sequence of SEQ ID NO: 1 or 2, or a sequence having identityto SEQ ID NO: 1 or 2. Typically it will have at least 75% identity toSEQ ID NO: 1 or 2 e.g., at least 80%, at least 85%, at least 90%, atleast 95%, at least 97%, at least 98%, at least 99%, identity to SEQ IDNO:1 or 2. The sequence may be found naturally in RSV.

Where the invention uses an ectodomain of F protein, in whole or inpart, it may comprise:

(i) a polypeptide comprising about amino acid 22-525 of SEQ ID NO: 1.

(ii) a polypeptide comprising about amino acids 23-525 of SEQ ID NO: 2.

(iii) a polypeptide comprising an amino acid sequence having at least75% identity (e.g., at least 80%, at least 85%, at least 90%, at least95%, at least 97%, at least 98%, at least 99% identity) to (i) or (ii).

(iv) a polypeptide comprising a fragment of (i), (ii) or (iii), whereinthe fragment comprises at least one F protein epitope. The fragment willusually be at least about 100 amino acids long, e.g., at least about150, at least about 200, at least about 250, at least about 300, atleast about 350, at least about 400, at least about 450 amino acidslong.

The ectodomain can be an F₀ form with or without the signal peptide, orcan comprises two separate peptide chains (e.g., an F₁ subunit and a F₂subunit) that are associated with each other, for example, the subunitsmay be linked by a disulfide bridge. Accordingly, all or a portion ofabout amino acid 101 to about 161, such as amino acids 110-136, may beabsent from the ectodomain. Thus the ectodomain, in whole or in part,can comprise:

(v) a first peptide chain and a second peptide chain that is associatedwith the first polypeptide chain, where the first peptide chaincomprises an amino acid sequence having at least 75% identity (e.g., atleast 80%, at least 85%, at least 90%, at least 95%, at least 97%, atleast 98%, at least 99%, or even 100% identity) to about amino acid 22to about amino acid 101 of SEQ ID NO: 1 or to about amino acid 23 toabout amino acid 101 of SEQ ID NO: 2, and the second peptide chaincomprises an amino acid sequence having at least 75% identity (e.g., atleast 80%, at least 85%, at least 90%, at least 95%, at least 97%, atleast 98%, at least 99%, or even 100% identity) to about amino acid 162to about 525 of SEQ ID NO: 1 or to about amino acid 162 to 525 of SEQ IDNO: 2.

(vi) a first peptide chain and a second peptide chain that is associatedwith the first polypeptide chain, where the first peptide chaincomprises an amino acid sequence comprising a fragment of about aminoacid 22 to about amino acid 101 of SEQ ID NO: 1 or of about amino acid23 to about amino acid 109 of SEQ ID NO: 2, and the second peptide chaincomprises a fragment of about amino acid 162 to about amino acid 525 ofSEQ ID NO: 1 or of about amino acid 161 to about amino acid 525 of SEQID NO: 2. One or both of the fragments will comprises at least one Fprotein epitope. The fragment in the first peptide chain will usually beat least 20 amino acids long, e.g., at least 30, at least 40, at least50, at least 60, at least 70, at least 80 amino acids long. The fragmentin the second peptide chain will usually be at least 100 amino acidslong, e.g., at least 150, at least 200, at least 250, at least 300, atleast 350, at least 400, at least 450 amino acids long.

(vii) a molecule obtainable by furin digestion of (i), (ii), (iii) or(iv).

Thus an amino acid sequence used with the invention may be foundnaturally within RSV F protein (e.g., a soluble RSV F protein lacking TMand CT, about amino acids 522-574 of SEQ ID NOS: 1 or 2), and/or it mayhave one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30) singleamino acid mutations (insertions, deletions or substitutions) relativeto a natural RSV sequence. For instance, it is known to mutate Fproteins to eliminate their furin cleavage sequences, thereby preventingintracellular processing. In certain embodiments, the RSV F proteinlacks TM and CT (about amino acids 522-574 of SEQ ID NOS: 1 or 2) andcontains one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30)single amino acid mutations (insertions, deletions or substitutions)relative to a natural RSV sequence.

Furin-Cleavage, Trypsin-Cleavage and Fusion Peptide Mutations

RSV F polypeptides or proteins may contain one or more mutations thatprevent cleavage at one or both of the furin cleavage sites (i.e., aminoacids 109 and 136 of SEQ ID NOS: 1 and 2). These mutations can preventaggregation of the soluble polypeptides or proteins and therebyfacilitate purifications, can prevent cell-cell fusion if the RSV Fprotein is expressed on the surface of a cell, such as by expressionfrom a viral replicon (e.g., alphavirus replicon particles), or if theRSV F protein is a component of a virus-like particle. These mutations,alone or in combination with other mutations described herein, may alsostabilize the protein in the pre-fusion conformation.

Examples of suitable furin cleavage mutations include replacement ofamino acid residues 106-109 of SEQ ID NO: 1 or 2 with RARK (SEQ IDNO:77), RARQ (SEQ ID NO:78), QAQN (SEQ ID NO:79), or IEGR (SEQ IDNO:80). Alternatively, or in addition, amino acid residues 133-136 ofSEQ ID NO: 1 or 2 can be replaced with RKKK (SEQ ID NO:81), ΔΔΔR, QNQN(SEQ ID NO:82), QQQR (SEQ ID NO:83) or IEGR (SEQ ID NO:80). (Δ indicatesthat the amino acid residue has been deleted.) These mutations can becombined, if desired, with other mutations described herein, such asmutations in the p27 region (amino acids 110-136 of SEQ ID NOS: 1 or 2),including deletion of the p27 region in whole or in part.

These furin cleavage mutations can be combined, if desired, with othermutations described herein, such as trypsin cleavage mutations andfusion peptide mutations. Examples of suitable trypsin cleavagemutations include deletion of any lysine or arginine residue betweenabout position 101 and position 161 of SEQ ID NO:1 or 2, or replacementof any such lysine or arginine residue with an amino acid other thanlysine or arginine. For example, lysine and/or arginine residues in thep27 region (about amino acids 110-136 of SEQ ID NOS: 1 or 2) can besubstituted or deleted, including deletion of the p27 region in whole orin part.

Alternatively or in addition to the furin-cleavage mutations, RSV Fpolypeptides or proteins may contain one or more mutations in the fusionpeptide region (amino acids 137 and 153 of SEQ ID NOS: 1 or 2). Forexample, this region can be deleted in whole or in part.

In particular embodiments, the sequence of amino acid residue 100-150 ofthe RSV F polypeptide or protein, such as SEQ ID NO:1, SEQ ID NO:2, orthe soluble ecto domains thereof, is

(Furmt) (SEQ ID NO: 3)TPATNNRARKELPRFMNYTLNNAKKTNVTLSKKRKKKFLGFLLGVGSAIAS (Furdel)(SEQ ID NO: 4) TPATNNRARQELPRFMNYTLNNAKKTNVTLSKKRFLGFLLGVGSAIAS (Furx)(SEQ ID NO: 6) TPATNNQAQNELPRFMNYTLNNAKKTNVTLSQNQNQNFLGFLLGVGSAIAS(Furx R113Q, K123N, K124N) (SEQ ID NO: 5)TPATNNQAQNELPQFMNYTLNNANNTNVTLSQNQNQNFLGFLLGVGSAIAS(Furx R113Q, K123Q, K124Q)) (SEQ ID NO: 92)TPATNNQAQNELPQFMNYTLNNAQQTNVTLSQNQNQNFLGFLLGVGSAIAS (Delp21Furx)(SEQ ID NO: 7) TPATNNQAQN-----------------------QNQNQNFLGFLLGVGSAI AS(Delp23Furx) (SEQ ID NO: 8) TPATNNQAQNQNQNFLGFLLGVGSAIAS (Delp21 furdel)(SEQ ID NO: 109) TPATNNRARQ-----------------------QNQQQRFLGFLLGVGSAI AS(Delp23furdel) (SEQ ID NO: 9)TPATNNRARQ-----------------------QQQRFLGFLLGVGSAIAS (Nterm Furin)(SEQ ID NO: 10) TPATNNRARRELPQFMNYTLNNAQQTNVTLSQNQNQNFLGFLLGVGSAIAS(Cterm Furin) (SEQ ID NO: 11)TPATNNQAQNELPQFMNYTLNNAQQTNVTLSKKRKRRFLGFLLGVGSAIAS(Fusion peptide deletion 1) (SEQ ID NO: 12)TPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRR----------- SAIAS,(Fusion peptide deLetion 2) (SEQ ID NO: 91)TPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRR--------- GVGSAIAS, or (Factor Xa)(SEQ ID no: 13) TPATNNIEGRELPRFMNYTLNNAKKTNVTLSKKIEGRFLGFLLGVGSAIAS;wherein the symbol “-” indicates that the amino acid at that position isdeleted.

In addition to furin-cleavage and fusion peptide mutations, oralternatively, soluble RSV F polypeptides or proteins, such as thosethat lack the transmembrane region and cytoplasmic tail, may contain oneor more oligomerization sequences. When an oligomerization sequence ispresent, it is preferably a trimerization sequence. Suitableoligomerization sequences are well known in the art and include, forexample, the coiled coil of the yeast GCN4 leucine zipper protein,trimerizing sequence from bacteriophage T4 fibritin (“foldon”), and thetrimer domain of influenza HA. These and other suitable oligomerizationsequences are described in greater detail herein.

In particular embodiments, the sequence of the carboxy terminus of theRSV F polypeptide or protein, starting from position 480, is

(GCN) (SEQ ID NO: 14)PLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNDKIEEILSKIYHI ENEIARIKKLIGE  (HA)(SEQ ID NO: 15) PLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNEKFHQIEKEFSEVEGRIQDLEK (Idealized helix) (SEQ ID NO: 16)PLVFPSDEFDASISQINEKINQILAFIRKIDELLHNIN (foldon short) (SEQ ID NO: 17)PLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNGSGYIPEAPRDGQ AYVRKDGEWVLLSTFL; or(foldon long) (SEQ ID NO: 18)PLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNNKNDDKGSGYIPEAPRDGQAYVRKDGEWVLLSTFL

In addition to any combination of furin-cleavage mutations, fusionpeptide mutations and added oligomerization sequences, or alternatively,RSV F polypeptides or proteins that contain a transmembrane region maycontain an added amino acid sequence that provides a protease cleavagesite. This type of RSV F polypeptide or protein can be produced byexpression on the surface of a cell, and recovered in soluble form aftercleavage from the cell surface using an appropriate protease. Generally,the amino acid sequence that provides a protease cleavage site will belocated within about 60 amino acids, about 50 amino acids, about 40amino acids, about 30 amino acids, about 20 amino acids, about 10 aminoacids, or substantially adjacent to the amino terminus of thetransmembrane domain (amino acid 525 of SEQ ID NO:1 or 2). Many suitableamino acid sequences that are cleaved by commercially availableproteases are well-known in the art. For example, thrombin cleaves thesequence LVPR (SEQ ID NO:75), factor Xa cleaves the sequence IEGR andenterokinase cleaves the sequence DDDDK (SEQ ID NO:76). These amino acidsequences can be introduced into an RSV F polypeptide. In particularembodiments, the sequence of the RSV F polypeptide or protein, startingfrom position 488 to the TM region is a sequence shown in FIG. 2.

Immunogenic polypeptides used according to the invention will usually beisolated or purified. Thus, they will not be associated with moleculeswith which they are normally, if applicable, found in nature. Forexample, an F protein used with the invention will not be in the form ofa RSV virion (although it may be in the form of an artificial virion,such as a virosome or VLP).

Polypeptides will usually be prepared by expression in a recombinanthost system. Generally, they (e.g., RSV ecto-domains) are produced byexpression of recombinant constructs that encode the ecto-domains insuitable recombinant host cells, although any suitable methods can beused. Suitable recombinant host cells include, for example, insect cells(e.g., Aedes aegypti, Autographa californica, Bombyx mori, Drosophilamelanogaster, Spodoptera frugiperda, and Trichoplusia ni), mammaliancells (e.g., human, non-human primate, horse, cow, sheep, dog, cat, androdent (e.g., hamster), avian cells (e.g., chicken, duck, and geese),bacteria (e.g., E. coli, Bacillus subtilis, and Streptococcus spp.),yeast cells (e.g., Saccharomyces cerevisiae, Candida albicans, Candidamaltosa, Hansenual polymorpha, Kluyveromyces fragilis, Kluyveromyceslactis, Pichia guillerimondii, Pichia pastoris, Schizosaccharomycespombe and Yarrowia lipolytica), Tetrahymena cells (e.g., Tetrahymenathermophila) or combinations thereof. Many suitable insect cells andmammalian cells are well-known in the art. Suitable insect cellsinclude, for example, Sf9 cells, Sf21 cells, Tn5 cells, Schneider S2cells, and High Five cells (a clonal isolate derived from the parentalTrichoplusia ni BTI-TN-5B1-4 cell line (Invitrogen)). Suitable mammaliancells include, for example, Chinese hamster ovary (CHO) cells, humanembryonic kidney cells (HEK293 cells, typically transformed by shearedadenovirus type 5 DNA), NIH-3T3 cells, 293-T cells, Vero cells, HeLacells, PERC.6 cells (ECACC deposit number 96022940), Hep G2 cells, MRC-5(ATCC CCL-171), WI-38 (ATCC CCL-75), fetal rhesus lung cells (ATCCCL-160), Madin-Darby bovine kidney (“MDBK”) cells, Madin-Darby caninekidney (“MDCK”) cells (e.g., MDCK (NBL2), ATCC CCL34; or MDCK 33016, DSMACC 2219), baby hamster kidney (BHK) cells, such as BHK21-F, HKCC cells,and the like. Suitable avian cells include, for example, chickenembryonic stem cells (e.g., EBx® cells), chicken embryonic fibroblasts,chicken embryonic germ cells, duck cells (e.g., AGE1.CR and AGE1.CR.pIXcell lines (ProBioGen) which are described, for example, in Vaccine27:4975-4982 (2009) and WO2005/042728), EB66 cells, and the like.

Suitable insect cell expression systems, such as baculovirus systems,are known to those of skill in the art and described in, e.g., Summersand Smith, Texas Agricultural Experiment Station Bulletin No. 1555(1987). Materials and methods for baculovirus/insert cell expressionsystems are commercially available in kit form from, inter alia,Invitrogen, San Diego Calif. Avian cell expression systems are alsoknown to those of skill in the art and described in, e.g., U.S. Pat.Nos. 5,340,740; 5,656,479; 5,830,510; 6,114,168; and 6,500,668; EuropeanPatent No. EP 0787180B; European Patent Application No. EP03291813.8; WO03/043415; and WO 03/076601. Similarly, bacterial and mammalian cellexpression systems are also known in the art and described in, e.g.,Yeast Genetic Engineering (Barr et al., eds., 1989) Butterworths,London.

Recombinant constructs encoding RSV F protein ecto-domains can beprepared in suitable vectors using conventional methods. A number ofsuitable vectors for expression of recombinant proteins in insect ormammalian cells are well-known and conventional in the art. Suitablevectors can contain a number of components, including, but not limitedto one or more of the following: an origin of replication; a selectablemarker gene; one or more expression control elements, such as atranscriptional control element (e.g., a promoter, an enhancer, aterminator), and/or one or more translation signals; and a signalsequence or leader sequence for targeting to the secretory pathway in aselected host cell (e.g., of mammalian origin or from a heterologousmammalian or non-mammalian species). For example, for expression ininsect cells a suitable baculovirus expression vector, such as pFastBac(Invitrogen), is used to produce recombinant baculovirus particles. Thebaculovirus particles are amplified and used to infect insect cells toexpress recombinant protein. For expression in mammalian cells, a vectorthat will drive expression of the construct in the desired mammalianhost cell (e.g., Chinese hamster ovary cells) is used.

RSV F protein ecto-domain polypeptides can be purified using anysuitable methods. For example, methods for purifying RSV F ecto-domainpolypeptides by immunoaffinity chromatography are known in the art.Ruiz-Arguello et al., J. Gen. Virol., 85:3677-3687 (2004). Suitablemethods for purifying desired proteins including precipitation andvarious types of chromatography, such as hydrophobic interaction, ionexchange, affinity, chelating and size exclusion are well-known in theart. Suitable purification schemes can be created using two or more ofthese or other suitable methods. If desired, the RSV F proteinecto-domain polypeptides can include a “tag” that facilitatespurification, such as an epitope tag or a HIS tag. Such taggedpolypeptides can conveniently be purified, for example from conditionedmedia, by chelating chromatography or affinity chromatography.

The RSV F polypeptides may also be produced in situ by expression ofnucleic acids that encode them in the cells of a subject. For example,by expression of a self-replicating RNA described herein.

Polypeptides may include additional sequences in addition to the RSVsequences. For example, a polypeptide may include a sequence tofacilitate purification (e.g., a poly-His sequence). Similarly, forexpression purposes, the natural leader peptide of F protein may besubstituted for a different one. For example, reference 6 used ahoneybee melittin leader peptide in place of the natural one.

Form and Conformation of Polypeptides

The invention includes immunogenic compositions that include any of theforms and conformations of RSV F polypeptides and proteins disclosedherein, including any desired combination of the forms and conformationsof RSV F polypeptides and proteins disclosed herein. The RSV Fpolypeptide can be a monomer, or the RSV F protein can be a trimercomprising three monomer polypeptides. Trimers can be monodispersed orcan be in the form of a rosette, for example, due to interactionsbetween the fusion peptides of individual timers. Immunogeniccompositions may comprise polypeptides that are monomers, trimers, acombination of monomers and trimers (e.g., in dynamic equilibrium),rosettes of trimers, and any combination of the foregoing. In addition,as described further herein, the RSV F protein can be in a post-fusionconformation, a pre-fusion conformation, or intermediate conformation.

The RSV F protein can be in a pre-fusion conformation, a post-fusionconformation or an intermediate conformation. The “post fusionconformation” of RSV F protein is believed to be the low energyconformation of native RSV F, and is a trimer characterized by thepresence of a six-helix bundle comprising 3 HRB and 3HRA regions. Thepost-fusion conformation has a characteristic “crutch” or “golf tee”shape by electron microscopy. The “pre-fusion conformation” of RSV Fprotein is a conformation characterized by a trimer that contains acoiled coil comprising 3 HRB regions. The fusion peptide is not exposedin the pre-fusion conformation and, therefore, prefusion conformationsgenerally do not form rosettes, and have a “lollipop” or “ball and stem”shape by electron microscopy.

In some aspects, the RSV F protein is in the post-fusion conformation.For example, the RSV F protein can be in the form of a monodispersetrimer in the post-fusion conformation, or in the form of a rosettecomprised of post-fusion trimers.

In some embodiments, the RSV F polypeptide is a monomer. In someembodiments, the RSV F polypeptide is a trimer.

In other aspects, the RSV F protein is in the pre-fusion conformation.Without wishing to be bound by any particular theory, it is believedthat the pre-fusion conformation or intermediate forms of RSV F proteinmay contain epitopes that are the same as those on the RSV F proteinexpressed on natural RSV virions, and therefore provide advantages foreliciting neutralizing antibodies.

Some aspects of the invention use a polypeptide that disfavors thepost-fusion conformation of the F protein. Preferably, the polypeptides(in whole or in part) will display an epitope of the pre-fusion Fprotein or an epitope of an intermediate conformation in the conversionfrom the pre-fusion conformation to the post-fusion conformation. Thesepolypeptides may be native or mutated F proteins in a pre-fusion state,may be native or mutated F proteins in an intermediate conformation, ormay be a population of native or mutated proteins where the post-fusionconformation has been disfavored or preferentially excluded. In certaininstances the native or mutated protein may be combined with one or moreadditional molecules that assist in maintaining the polypeptides in oneof the foregoing states such as a monoclonal antibody thatpreferentially binds the pre-fusion conformation or an intermediateconformation. In addition, the polypeptides may be derivatives of nativeF proteins. Such derivatives include polypeptides comprising one or morefragments of a native F protein, fusion polypeptides comprising a nativeF protein (or fragment thereof) and a heterologous sequence, andpolypeptides comprising a native F protein sequence having one or moremutations. These (or other) modifications may disfavor the post-fusionconformation. Exemplary approaches to disfavor the post-fusionconformation include stabilizing the pre-fusion conformation,stabilizing an intermediate conformation, destabilizing the post-fusionconformation or increasing the activation barrier of one or more stepsleading to the post fusion conformation.

In another embodiment, the invention is a polypeptide that displays atleast one epitope that is specific to the pre-fusion conformation Fprotein or an intermediate conformation F protein. An epitope that isspecific to the pre-fusion conformation F protein or an intermediateconformation F protein is an epitope that is not presented in thepost-fusion conformation. It is preferred that the at least one epitopeis stably presented, e.g., the epitope is stably presented in solutionfor at least twelve hours, at least one day, at least two days, at leastfour days, at least six days, at least one week, at least two weeks, atleast four weeks, or at least six weeks.

Such polypeptides may be native or mutated F proteins in the pre-fusionstate, an intermediate state or a population of states where thepost-fusion state is underrepresented or at a lower percentage than forisolated native F proteins, or it may be a derivative of a native Fprotein. Such derivatives include polypeptides comprising one or morefragments of a native F protein, fusion polypeptides comprising a nativeF protein (or fragment thereof) and a heterologous sequence, andpolypeptides comprising a native F protein sequence having one or moremutations. These (or other) modifications may stabilize an F proteinamino acid sequence in its pre-fusion conformation, stabilize an Fprotein amino acid sequence in an intermediate conformation, destabilizethe post fusion conformation of an F protein amino acid sequence,increase the energy barrier in a transition leading to the post-fusionconformation of an F protein amino acid sequence, or a combination oftwo or more of the foregoing.

The TM and/or CT domains of F proteins are important for the stabilityof the pre-fusion conformation (8). Thus these domains may usefully beretained in immunogens of the invention. As it may be desirable not toinclude transmembrane domains in soluble immunogens, though, thefunctional effect of TM may be achieved by other means. For instance,the pre- and post-fusion behavior of F protein of parainfluenza virus 5has been studied in some detail (6), and the authors stabilized the ED'spre-fusion structure by fusing a heterologous trimerization domain tothe C-terminus of the ED.

Oligomerization Domain

In another embodiment of the invention, the composition may comprise apolypeptide (e.g., recombinant polypeptide) that comprises a firstdomain and a second domain, wherein (i) the first domain comprises theRSV F protein (e.g. RSV ectodomain, in whole or part), and (ii) thesecond domain comprises a heterologous oligomerization domain. Thesecond domain permits oligomerization of the polypeptide, therebyfacilitating the first domain to adopt a pre-fusion state or anintermediate state. The polypeptide preferably will be present as anoligomer, and in particular as a trimer.

Various oligomerization domains are available to the skilled person.These are sequences of amino acids that can form a structure that caninteract with oligomerization domains (whether the same or different) inother polypeptides (whether the same or different) such that themultiple polypeptides can associate (usually non-covalently) to formoligomers, e.g., trimers. For instance, trimerization of the F proteinof HIV (i.e., gp160) has been achieved by fusing it to the catalyticsubunit of E. coli aspartate transcarbamoylase (ATCase), which isnaturally a stable trimer (9). Thus this subunit of ATCase may be usedwith the present invention. Similarly, trimerization of the F proteinsof HIV (10) and PIV5 (6) has been achieved by fusing their ectodomainsto GCNt. Thus the oligomerization domain used with the present inventionmay comprise the coiled coil of the yeast GCN4 leucine zipper protein(11). Trimerization of the ectodomain of HA protein from influenza Avirus has been achieved by using a trimerizing sequence (‘foldon’) fromthe bacteriophage T4 fibritin (GSGYIPEAPRDGQ AYVRKDGEWVLLSTFL—SEQ IDNO:19) (12). Thus the oligomerization domain used with the presentinvention may comprise such a foldon.

Naturally-occurring protein oligomers (both hetero-oligomers andhomo-oligomers) associate in a variety of different ways, e.g., byassociation of β-sheets in different monomers, by association ofα-helices in different monomers, by association of hydrophobic surfacepatches, etc. One common structural motif involved in proteinoligomerization is the coiled-coil domain. The coiled α-helix structuralmotif can itself form coils, and two, three, four or five α-helices canwrap around each other to form a left-handed super-helix known as the“coiled coil” though artificial right-handed super helices have beendesigned (13-19). The simplicity of the coiled-coil domain has made it apopular choice for designing chimeric proteins with definedoligomerization states (16).

In a coiled-coil structure the α-helices interact through hydrophobicresidues that form an apolar stripe along one side of each helix, andthere may also be stabilizing electrostatic interactions between sidechains on either side of this stripe. Within the abcdefg heptad repeatof an α-helix, the apolar stripe is defined by hydrophobic side chainsat residues a and d, with any electrostatic interactions being primarilyat residues e and g. Position a is most frequently Leu, Ile or Ala andposition d is usually Leu or Ala. Residues e and g are often Glu or Gln,with Arg and Lys also prominent at position g. Charged residues arecommon at positions b, c and f as these residues are in contact withsolvent. There are exceptions to this general heptad pattern, however,and Pro residues are sometimes found within the heptad. Such exceptionsusually have functional significance including, by way of example,destabilization of the oligomerization domain to allow refolding andrearrangement such as occurs in the F protein.

Hundreds of coiled-coil domain sequences are known in the art, and anysuitable sequence can be used as an oligomerization domain with theinvention, provided that it retains the ability to oligomerize withother coiled-coil domains and that it does not destroy the function ofthe other domains within the polypeptide. It is preferred to use acoiled-coil domain which is found extracellularly (20) and whichnaturally acts as an oligomerization domain. As an alternative to usinga natural coiled-coil domain, artificial coiled-coil domains can be used(21, 22). Owing to the highly repetitive structure of a coiled-coildomain, the domain is particularly amenable to computer modeling as thebackbone portions of each amino acid residue may be parameterized ratherthan treating each backbone portion of a residue as a unique unit withits own variables. Domain (b) may include a leucine zipper sequence oran alanine zipper sequence (23).

The coiled-coil domain used in the polypeptide of the invention ispreferably one which forms a trimer, such that the polypeptide of theinvention can also assemble into a trimer. Preferred coiled-coil domainsare those taken from bacterial transmembrane proteins. A preferredsubset of transmembrane proteins is the adhesins (i.e., cell-surfaceproteins that mediate adhesion to other cells or to surfaces), andparticularly non-fimbrial adhesins (e.g., in the oligomerizationcoiled-coil adhesins, or ‘Oca’, family). Specific sequences for use withthe invention include those disclosed in reference 24 from Yersiniaenterocolitica adhesin YadA, Neisseria meningitidis adhesin NadA,Moraxella catarrhalis surface protein UspA2, and other adhesins, such asthe HadA adhesin from Haemophilus influenzae biogroup aegyptius etc (SEQID NOs 28-31 & 42-58 of ref. 24). In addition, the eukaryotic heat-shocktranscription factor has a coiled-coil trimerization domain that can beseparately expressed and therefore used with the invention.

Within the amino acid sequence of a polypeptide having a coiled-coilregion, the heptad-repeat nature of the α-helices means that theboundary of the coiled-coil domain can be determined with someprecision, but the precise residue where a coiled-coil arrangement canbe said to end may not be known with absolute accuracy. This lack ofabsolute precision is not a problem for practicing the invention,however, as routine testing can reveal whether the coiled-coil requiresany particular amino acid residue for which there might be doubt. Evenso, the invention does not require the boundaries to be known withabsolute precision, as the only basic requirement for the invention isthat the coiled-coil domain should function in a way that allows thepolypeptide to oligomerize with other coiled-coil domains withoutdestroying the function of the other domains within the polypeptide.

Another class of oligomerization domain that can be used with theinvention is found in the left-handed triple helix known as the collagenhelix (25). These triple helix-forming sequences involve a basictripeptide repeat sequence of ¹Gly-²Xaa-³Xaa, where ²Xaa is often Pro,and ³Xaa is often 4-hydroxyproline. Although this motif is known as the“collagen” helix, it is found in many proteins beyond just collagen. Theoligomerization domain may thus be a sequence comprising multiplerepeats of the sequence motif ¹Gly-²Xaa-³Xaa, which motif folds to forma helical structure that can oligomerize with corresponding helicalstructures in other polypeptide chains.

Collagen also provides another class of oligomerization domain.Reference 26 describes a motif found in the non-collagenous domain 1(NC1) of type X collagen, and this motif can be used for trimer andhigher order multimer formation without a triple helix. This trimericassociation is highly thermostable without intermolecular disulfidebonds. The oligomerization domain may thus comprise an NC1 sequence.

Other oligomerization domains may be derived from the transmembranedomains of oligomeric TM proteins. As these are usually lipophilic,hydrophobic residues positioned on the outside of their TM regions maybe substituted with charged residues, to provide a soluble domain. Suchmethods of solubilizing transmembrane domains by protein engineering areknown in the art, for example from reference 27. This method has alsobeen used for GCN4, where the “a” and “d” heptad repeat positions werereplaced with isoleucine (11): KQIEDKIEEILSKIYHIENEIARIKKLIGEA (SEQ IDNO: 20). Suitable coiled coil sequences for use within theoligomerization domain will usually be between 20 and 35 amino acidslong, e.g., 23 to 30 amino acid residues long.

Oligomerization domains used with the invention can generally maintainan oligomeric structure without the need for the formation ofinter-monomer disulfide bridges, but oligomers containingdisulfide-linked monomers are not excluded from the invention.

As an alternative, or in addition, to using an oligomerization domain tostabilize an F protein in its pre-fusion conformation, mutation can beused. For instance, reference 28 reports that mutation in a conservedregion of the F2 subunit of the F proteins in simian virus 5 or hendravirus can influence the stability of the pre-fusion conformation.

In some circumstances a low pH may also be used to favor the pre-fusionconformation.

Stabilization of the HRB Domain Trimer

In another preferred aspect of the present invention, the post-fusionconformation of the F protein may be disfavored by stabilization of theHRB domain trimer. The HRB domain forms a triple stranded coiled coil inthe pre-fusion and likely the intermediate forms. As discussed in thepreceding section, due to their simplicity, coiled-coils have beenextensively studied as model systems for intermolecular interactionsbetween proteins and as model systems for longer range intra-molecularinteractions (i.e., tertiary folding interactions). These studies areuseful in teaching methods that may be used to stabilize the HRB domainin the trimeric coiled-coil form. By way of example, one or moreresidues at the a and/or d positions of the heptad repeat may bereplaced with residues that favor formation of stable trimericcoiled-coils such as Ile residues. In addition, though less preferred,disfavorable ionic interactions at the e and g positions may be deletedor favorable ionic interactions at the e and g positions may be added.

The preferred region of the HRB domain for manipulation is the heptadrepeat between P484-N517. Preferred examples of a and d residues totarget for mutations are F488, I492, V495, I499, S502, I506, S509, L512,and V516. The serine residues are especially preferred as replacement ofthe hydrophilic residues with hydrophobic residues would stabilize thehydrophobic core of the coiled-coil. Another preferred target would bethe phenylalanine with a smaller hydrophobic residue that would packbetter in the core such as an isoleucine.

Destabilization of the HRA Domain Trimer

In another preferred aspect of the present invention, the post-fusionconformation of the F protein may be disfavored by destabilization ofthe HRA domain trimer. The HRA domain forms a triple stranded coiledcoil in the post-fusion and possibly one or more intermediate forms. Byway of example, one or more residues at the a and/or d positions of theheptad repeat may be replaced with residues that disfavor formation ofstable trimeric coiled-coils. In addition, though less preferred,favorable ionic interactions at the e and g positions may be deleted ordisfavorable ionic interactions at the e and g positions may be added.Preferably such mutations will be selected that have minimal impact onthe stability of the HRA domain in the pre-fusion conformation as may bemodeled based upon the available crystal structures of the PIV5 Fprotein in the pre-fusion and post fusion forms.

Other Modifications

In addition to the foregoing modifications, modifications can further bedesigned based upon molecular modeling of the hRSV F proteins based uponthe available crystal structures of the PIV5 F protein in the pre-fusionand post fusion forms. Mutations may be made that destabilize thepost-fusion conformation such as the 6HB fold of the HRA and HRB domainsor that stabilize the pre-fusion conformation such as the HRA fold inthe pre-fusion conformation. In addition, the energy barrier of thetransitions leading to the post-fusion conformation may be increased.While one of skill in the art will appreciate that stabilizing thestarting conformation or destabilizing the end conformation can have theeffect of increasing the energy barrier, other modifications that affectthe transition state itself may be introduced.

As an additional example, the amino acids N-terminal to the HRB domain(approximately a.a. 449-482, preferably V459-F483) act as a “tether”that allows the HRB domain to shift from one side of the F proteintrimer to the other side so that the HRB domain can participate in the6HB of the post-fusion conformation. Deletion of one or more of theseamino acids will impair or outright prevent the HRB domain fromparticipating in the 6HB fold of the post-fusion conformation of the Fprotein (see FIG. 3). In addition, the interaction between the tetherand the F protein in the pre-fusion conformation can be stabilized toprevent the tether from pulling away to allow the HRB domain toparticipate in the 6HB fold. Examples of stabilizing mutations thatcould be made are cysteine bridges between the tether and the portion ofthe F protein which the tether contacts in the pre-fusion conformation.

Yet another example is stabilization of the HRA in the pre-fusionconformation (residues T50-Y306). Again, based upon the crystalstructures of homologous F proteins, the hydrophobic core may bestabilized by replacing buried hydrophilic or ionic residues withsimilarly sized hydrophobic residues. Also, cysteine bridges may beintroduced at the surface or within the core. In addition, as wasdemonstrated with extensive crystal structure analysis of lysozymemutants, the hydrophobic cores or proteins are relatively rigid andtherefore introducing holes predictably destabilized the lysozymemutants. Similarly, repacking the core of the F protein in thepre-fusion conformation to eliminate any natural holes can stabilize theF protein in the pre-fusion or intermediate forms, thus disfavoring thepost-fusion conformation.

Methods for Preparing Compositions

The invention relates to methods for preparing compositions and tocompositions that contain RSV F protein, in particular soluble RSV Fecto-domain polypeptides, including immunogenic compositions.Preferably, the RSV F ecto-domain polypeptides are in a single form,such as uncleaved monomers, uncleaved trimers, cleaved trimers, rosettesof cleaved trimers, or in a dynamic equilibrium between a subset of suchforms (e.g., equilibrium between uncleaved monomers and uncleavedtrimers). The invention provides several advantages. For example, asdescribed herein, the invention provides methods for producingcompositions that contain a predominate desired form of RSV F protein,or a single desired form of RSV F protein, such as uncleaved monomers,uncleaved trimers, cleaved trimers, rosettes of cleaved trimers, adynamic equilibrium between a subset of such forms (e.g., equilibriumbetween uncleaved monomers and uncleaved trimers), or a mixture ofdesired form of RSV F protein. These types of compositions can be usedfor a variety of purposes, such as, in the production of immunogeniccompositions that can be used to produce vaccines. The presence of asingle desired form of RSV F, or a dynamic equilibrium between knownforms, in an immunogenic composition, provides for more predictableformulation, solubility and stability, and for a more predictable immuneresponse when the composition is administered to a subject.

When RSV F protein ecto-domain polypeptides are produced by conventionalrecombinant expression in host cells, the polypeptides are cleaved atthe furin cleavage sites at about position 109/110 and at about position136/137 during production in the host cell before they are secreted intothe culture media. Cleavage of the polypeptides by the host cells ispermissive for RSV F protein ecto-domain polypeptide refolding, whichresults in exposure of the hydrophobic fusion peptide. Accordingly, thecleaved RSV F protein ecto-domain polypeptides, due to the presence ofan exposed fusion peptide, form rosettes and associate with lipids andlipoproteins that are derived from the host cells and culture media. Infact, electron microscopy of cleaved RSV F ectodomain that are producedin insect cells and purified by virtue of a HIS₆-tag showed that thepolypeptides had a crutch shape consistent with a post-fusion form andwere bound to what appeared to residual cell debris. Accordingly, highpurity preparations of rosettes and other forms and confirmations of RSVF protein ecto-domain polypeptides cannot be readily obtained byconventional recombinant expression in host cells.

Methods for Producing Cleaved RSV F Protein Ecto-Domain Polypeptides

In one aspect, the invention is a method for preparing a compositionthat contains cleaved RSV F protein ecto-domain polypeptides. Ingeneral, the method involves providing uncleaved RSV F proteinecto-domain polypeptides and then cleaving them to produce a F₁ subunitand a F₂ subunit. As described herein, uncleaved RSV F proteinecto-domain polypeptides can be readily purified and separated fromcontaminating lipids and lipoproteins using suitable methods, such assize exclusion chromatography. Without wishing to be bound by anyparticular theory, it is believed that the hydrophobic fusion peptide isnot exposed in the uncleaved RSV F protein ecto-domain polypeptides and,therefore, the uncleaved polypeptides do not associate with lipid andlipoprotein contaminants. As further described herein, uncleaved RSV Fprotein ecto-domains can be cleaved to produce F₁ and F₂ subunits, whichcan be purified as trimers, rosettes of trimers, or a mixture of trimersand rosettes of trimers.

Uncleaved RSV F protein ecto-domain polypeptides can be produced usingany suitable method. For example, by recombinant production in hostcells that do not contain active furin or furin-like proteases at thetime the RSV F protein ecto-domain polypeptides are being produced. Avariety of methods can be used to achieve this method of production,such as, production in recombinant host cells that are mutated toprevent expression of furin or furin-like protease (conditionally orcomplete “knock-out”), and various methods that reduce or preventexpression of furin or furin-like proteases in the host cells, forexample, using RNA interference or other similar methods, or inhibitingfurin or furin-like protease activity in host cells using inhibitors ofthe proteases.

Uncleaved RSV F protein ecto-domain polypeptides are preferably producedby recombinant expression of constructs that encode a RSV F proteinecto-domain in which the amino acid sequence of the furin cleavage sitesare altered, so that the RSV F protein ecto-domain polypeptides aresecreted by a host cell that produces the polypeptides uncleaved. Theuncleaved RSV F protein ecto-domain polypeptides can be produced usingany suitable host cell, such as insect cells (e.g., Aedes aegypti,Autographa californica, Bombyx mori, Drosophila melanogaster, Spodopterafrugiperda, and Trichoplusia ni), mammalian cells (e.g., human,non-human primate, horse, cow, sheep, dog, cat, and rodent (e.g.,hamster), avian cells (e.g., chicken, duck, and geese, bacteria (e.g.,E. coli, Bacillus subtilis, and Streptococcus spp.), yeast cells (e.g.,Saccharomyces cerevisiae, Candida albicans, Candida maltosa, Hansenualpolymorpha, Kluyveromyces fragilis, Kluyveromyces lactis, Pichiaguillerimondii, Pichia pastoris, Schizosaccharomyces pombe and Yarrowialipolytica), Tetrahymena cells (e.g., Tetrahymena thermophila) orcombinations thereof. Many suitable insect cells and mammalian cells arewell-known in the art. Suitable insect cells include, for example, Sf9cells, Sf21 cells, Tn5 cells, Schneider S2 cells, and High Five cells (aclonal isolate derived from the parental Trichoplusia ni BTI-TN-5B1-4cell line (Invitrogen)). Suitable mammalian cells include, for example,Chinese hamster ovary (CHO) cells, human embryonic kidney cells (HEK293cells, typically transformed by sheared adenovirus type 5 DNA), NIH-3T3cells, 293-T cells, Vero cells, HeLa cells, PERC.6 cells (ECACC depositnumber 96022940), Hep G2 cells, MRC-5 (ATCC CCL-171), WI-38 (ATCCCCL-75), fetal rhesus lung cells (ATCC CL-160), Madin-Darby bovinekidney (“MDBK”) cells, Madin-Darby canine kidney (“MDCK”) cells (e.g.,MDCK (NBL2), ATCC CCL34; or MDCK 33016, DSM ACC 2219), baby hamsterkidney (BHK) cells, such as BHK21-F, HKCC cells, and the like. Suitableavian cells include, for example, chicken embryonic stem cells (e.g.,EBx® cells), chicken embryonic fibroblasts, chicken embryonic germcells, duck cells (e.g., AGE1.CR and AGE1.CR.pIX cell lines (ProBioGen)which are described, for example, in Vaccine 27:4975-4982 (2009) andWO2005/042728), EB66 cells, and the like.

Suitable insect cell expression systems, such as baculovirus systems,are known to those of skill in the art and described in, e.g., Summersand Smith, Texas Agricultural Experiment Station Bulletin No. 1555(1987). Materials and methods for baculovirus/insert cell expressionsystems are commercially available in kit form from, inter alia,Invitrogen, San Diego Calif. Avian cell expression systems are alsoknown to those of skill in the art and described in, e.g., U.S. Pat.Nos. 5,340,740; 5,656,479; 5,830,510; 6,114,168; and 6,500,668; EuropeanPatent No. EP 0787180B; European Patent Application No. EP03291813.8; WO03/043415; and WO 03/076601. Similarly, bacterial and mammalian cellexpression systems are also known in the art and described in, e.g.,Yeast Genetic Engineering (Barr et al., eds., 1989) Butterworths,London.

Generally, the amino acid sequence of an uncleaved RSV F proteinecto-domain is altered to prevent cleavage at the furin cleavage sitesat about position 109/110 and about position 136/137, but contains anaturally occurring or introduced protease cleavage site, that whencleaved produce a F₁ subunit and a F₂ subunit. For example, theuncleaved RSV F protein ecto-domain polypeptide can have an amino acidsequence that is altered to prevent cleavage at the furin cleavage sitesat about position 109/110 and about position 136/137, but contain one ormore naturally occurring or introduced protease cleavage sites fromabout position 101 to about position 161.

A variety of particular amino acid sequences that will allow uncleavedRSV F protein ecto-domain polypeptides to be produced and expressed byhost cells, including amino acid sequences that are not cleaved at thefurin cleavage sites at about position 109/110 and about position136/137 can be readily designed and envisioned by a person of ordinaryskill in the art. In general, one or more amino acids that are part of,or are located near by, the furin cleavage sites at about position109/110 and about position 136/137 are independently replaced ordeleted. Some amino acid substitutions and deletions that are suitableto prevent cleavage of RSV F protein ecto-domain polypeptides are known.For example, the substitutions R108N, R109N, R108N/R109N, which inhibitcleavage at 109/110, and the substitution K131Q or the deletion of theamino acids at positions 131-134, which inhibit cleavage at 136/137,have been described Gonzalez-Reyes et al., Proc. Natl. Acad. Sci. USA,98:9859-9864 (2001). An uncleaved RSV F ecto-domain polypeptide thatcontains the amino acid substitutionsR108N/R109N/K131Q/R133Q/R135Q/R136Q has been described. Ruiz-Arguello etal., J. Gen. Virol. 85:3677687 (2004). As described in detail herein,additional RSV F protein amino acid sequences that result in the RSV Fecto-domain polypeptide being secreted from a host cell uncleavedcontain altered furin cleavage sites, e.g., alter amino acid sequencesat about positions 106-109 and at about positions 133-136. The alteredfurin cleavage sites contain at least one amino acid substitution ordeletion at about positions 106-109, and at least one amino acidsubstitution or deletion at about positions 133-136.

Similarly, a variety of particular amino acid sequences of uncleaved RSVF protein ecto-domain polypeptides that contain a protease cleavage site(e.g., naturally occurring or introduced) that when cleaved produce afirst subunit that comprises an F₁ and a second subunit that comprisesF₂, are possible and can be readily designed and envisioned. Forexample, the amino acid sequence of RSV F protein from about position101 to about position 161 contains trypsin cleavage sites, and one ormore of the trypsin cleavage sites can be cleaved by trypsin to generateF₁ and F₂ subunits. If desired, one or more suitable proteaserecognition sites can be introduced into the uncleaved RSV F proteinecto-domain polypeptide, for example, between about positions 101 toabout position 161. The introduced protease recognition sites can becleaved using the appropriate protease to generate F₁ and F₂ subunits.When a protease recognition site is introduced into the amino acidsequence of an uncleaved RSV F protein ecto-domain polypeptide, it ispreferred that the site is recognized by a protease that does not cleavethe ecto-domain of naturally occurring RSV F protein.

The method of this aspect of the invention includes: a) providinguncleaved RSV F protein ecto-domain polypeptides containing a proteasecleavage site that, when cleaved, produces F₁ and F₂ subunits, and b)cleaving the uncleaved RSV F protein ecto-domain polypeptides with aprotease that recognizes the protease cleavage site. In general, theamino acid sequence of the uncleaved RSV F protein ecto-domainpolypeptides contains altered furin cleavage sites, and the RSV Fprotein ecto-domain polypeptides are secreted from a host cell thatproduces them uncleaved at the furin cleavage sites at about positions106-109 and about positions 131-136.

The provided uncleaved RSV F protein ecto-domain polypeptides can bepurified to the desired degree. For example, the provided uncleaved RSVF protein ecto-domain polypeptides can be provided as a cell lysate,cell homogenate or cell culture conditioned media that is substantiallyunprocessed (e.g., unprocessed, or clarified only), or in partially orsubstantially purified form. In particular examples, the provideduncleaved RSV F protein ecto-domain polypeptides are provided in cellculture conditioned media selected from the group consisting of insectcell conditioned media, mammalian cell conditioned media, avian cellconditioned media, yeast cell conditioned media, Tetrahymena cellconditioned media, and combinations thereof.

It is generally preferred that the provided uncleaved RSV F proteinecto-domain polypeptides are purified, for example, purified to be atleast about 80%, at least about 85%, at least about 90%, at least about95% or substantially homogenous. As described herein, uncleaved RSV Fprotein ecto-domain polypeptides can be readily purified from lipids andlipoproteins, while conventionally produced cleaved forms of RSV Fprotein co-purify with lipid and lipoprotein contaminants. Accordingly,when purified uncleaved RSV F protein ecto-domain polypeptides areprovided, the method can be used to readily produce a compositioncontaining cleaved RSV F protein ecto-domains that are substantiallyfree of lipids or lipoproteins.

Suitable methods for cleaving polypeptides using a protease arewell-known and conventional in the art. Generally, the polypeptides tobe cleaved are combined with a sufficient amount of protease underconditions (e.g., pH, polypeptide and protease concentration,temperature) suitable for cleavage of the polypeptide. Many suitableproteases are commercially available, and suitable conditions forperforming polypeptide cleavage are well-known for many proteases. Ifdesired, the cleaved RSV F protein ecto-domain polypeptides can bepurified following cleavage with protease.

In one example of the method, uncleaved RSV F protein ecto-domainpolypeptides are provided that contain an intact fusion peptide, such asan uncleaved RSV F protein ecto-domain polypeptide in which none of theamino acids from positions 137-154 are substituted or deleted. In someembodiments, the provided uncleaved RSV F protein ecto-domainpolypeptides are purified. The provided uncleaved RSV F proteinecto-domain polypeptides that contain an intact fusion peptide arecleaved, and cleavage results in the formation of rosettes of cleavedRSV F protein ecto-domain polypeptide trimers. If desired, the rosettescan be purified further using any suitable methods, such as sizeexclusion chromatography.

In another example of the method, uncleaved RSV F protein ecto-domainpolypeptides are provided that contain an altered fusion peptide, suchas an uncleaved RSV F protein ecto-domain polypeptide in which aboutamino 137-152, about amino acids 137-153, about amino acids 137-145 orabout amino acids 137-142 are deleted. Other suitable fusion peptidedeletions have also been described, such as the deletion of the aminoacids at positions 137-146. Ruiz-Arguello et al., J. Gen. Virol.,85:3677-3687 (2004).

In some embodiments, the provided uncleaved RSV F protein ecto-domainpolypeptides are purified. The provided uncleaved RSV F proteinecto-domain polypeptides are cleaved, and cleavage results in theformation of trimers of cleaved RSV F protein ecto-domain polypeptides.If desired, the trimers can be purified further using any suitablemethods, such as size exclusion chromatography.

In particular examples of the method, the provided uncleaved RSV Fprotein ecto-domain polypeptides contain at least one polypeptideselected from the group consisting of furdel and delp23 furdel (e.g.,homogenous trypsin-cleavable furdel, homogenous trypsin-cleavable delp23furdel, or a mixture of trypsin-cleavable furdel and trypsin-cleavabledelp23 furdel). The provided uncleaved RSV F protein ecto-domainpolypeptides are cleaved, for example with trypsin, and cleavage resultsin the formation of cleaved trimers, rosettes of cleaved trimers, or acombination of cleaved trimers and rosettes of cleaved trimers of RSV Fprotein ecto-domain polypeptides. If desired, the cleaved trimers and/orrosettes of cleaved trimers can be purified further using any suitablemethods, such as size exclusion chromatography.

Methods for Producing Uncleaved RSV F Protein Ecto-Domain Polypeptides

In another aspect, the invention is a method for preparing a compositionthat contains uncleaved RSV F protein ecto-domain polypeptides. Ingeneral, the method involves providing a biological material thatcontains uncleaved RSV F protein ecto-domain polypeptides, such as acell lysate, cell homogenate or cell culture conditioned medium, andthen purifying the uncleaved RSV F protein ecto-domain polypeptides. Asdescribed herein, it has been discovered that purified uncleaved RSV Fprotein ecto-domain polypeptide monomers can self associate to formuncleaved trimers, and that there is a mixture of uncleaved monomers anduncleaved trimers or an equilibrium between the uncleaved monomers anduncleaved trimers. Without wishing to be bound by any particular theory,it is believed that the equilibrium favors the monomer, but that theequilibrium will shift toward the trimer in concentrated solutions.

The method of this aspect of the invention includes: a) providing abiological material that contains uncleaved RSV F protein ecto-domainpolypeptides, such as a cell lysate, cell homogenate or cell cultureconditioned medium; and b) purifying uncleaved RSV F protein ecto-domainpolypeptide monomers, trimers or a combination of monomers and trimersfrom the biological material. In some embodiments, uncleaved RSV Fprotein ecto-domain polypeptide monomers are purified, or uncleaved RSVF protein ecto-domain polypeptide trimers are purified, or monomers andtrimers are purified.

In general, the amino acid sequence of the uncleaved RSV F proteinecto-domain polypeptides contains altered furin cleavage sites, and theRSV F protein ecto-domain polypeptides are secreted from a host cellthat produces them uncleaved between about position 101 to aboutposition 161 (including at the furin cleavage sites at positions 106-109and 131-136). In more particular examples, the biological material thatcontains uncleaved RSV F protein ecto-domain polypeptides; includes atleast one polypeptide selected from the group consisting of furmt,furdel, delp21 furx, delp23 furx, delp21 furdel, delp23 furdel, and thefactor Xa construct, which can be cleaved using factor Xa.

In some embodiments, the amino acid sequence of the RSV F proteinecto-domain polypeptide contains altered furin cleavage sites, and otherprotease cleavage sites (e.g., trypsin cleavage sites) between aboutposition 101 and about position 161 are altered or deleted to preventprotease (e.g., trypsin) cleavage. For example, trypsin is well-known tocleave after lysine and arginine residues. In certain preferredembodiments, the amino acid sequence of the uncleaved RSV F proteinecto-domain polypeptide contains altered furin cleavage sites, one ormore lysine and/or arginine residues (e.g., all lysine and arginineresidues) present between about position 101 and about position 161 aredeleted or replaced with an amino acid that is not lysine or arginine,the RSV F protein ecto-domain polypeptides are secreted from a host cellthat produces them uncleaved between about position 101 and aboutposition 161, and the RSV F protein ecto-domain polypeptides are notcleaved by trypsin between about position 101 and about position 161.Preferably, the RSV F protein ecto-domain polypeptides are not cleavedby trypsin when a 1 mg/ml solution of RSV F protein ecto-domainpolypeptide (diluted in 25 mM Tris pH 7.5, 300 mM NaCl) is treated withone-one thousandth volume of trypsin solution (trypsin from bovineplasma diluted to a 1 mg/ml concentration in 25 mM Tris pH 7.5, 300 mMNaCl; final mass ratio in digestion reaction is 0.001:1 trypsin:RSV Fecto-domain; trypsin used at 10-15 BAEE units per mg protein) for 1 hourat 37° C.

If desired, the uncleaved RSV F protein ecto-domain polypeptides (e.g.,the polypeptides that contain alter furin cleavage sites, andpolypeptide that contain altered furin cleavage sites and alteredtrypsin cleavage sites) can further contain an altered fusion peptide,such as an uncleaved RSV F protein ecto-domain polypeptide in which, forexample, about amino acids 137-152 are deleted, about amino acids137-154 are deleted, about amino acids 137-145 are deleted or aboutamino acids 137-142 are deleted. Other suitable fusion peptide deletionshave also been described, such as the deletion of the amino acids atpositions 137-146. Ruiz-Arguello et al., J. Gen. Virol., 85:3677-3687(2004).

In particular embodiments, the method includes: a) providing abiological material that contains uncleaved RSV F protein ecto-domainpolypeptides, such as a cell lysate, cell homogenate or cell cultureconditioned medium, wherein the amino acid sequence of the uncleaved RSVF protein ecto-domain polypeptide contains altered furin cleavage sites,the lysine and arginine residues present between about position 101 andabout position 161 are deleted or replaced with an amino acid that isnot lysine or arginine, the RSV F protein ecto-domain polypeptides aresecreted from a host cell that produces them uncleaved between aboutposition 101 and about position 161, and the RSV F protein ecto-domainpolypeptides are not cleaved by trypsin between about position 101 andabout position 161; and b) purifying uncleaved RSV F protein ecto-domainpolypeptide monomers, timers or a combination of monomers and trimersfrom the biological material.

In more particular examples, the biological material that containsuncleaved RSV F protein ecto-domain polypeptides; includes at least onepolypeptide selected from the group consisting of Furx, Furx R113Q K123NK124N, delp21 furx and delp23 furx.

In other particular embodiments, the method includes: a) providing abiological material that contains uncleaved RSV F protein ecto-domainpolypeptides in which the fusion peptide is mutated (e.g., at least aportion of the fusion peptide is deleted), such as a cell lysate, cellhomogenate or cell culture conditioned medium; and b) purifyinguncleaved RSV F protein ecto-domain polypeptides from the biologicalmaterial. The uncleaved RSV F protein ecto-domain polypeptide cancontain altered furin cleavage sites, and the RSV F protein ecto-domainpolypeptides are secreted from a host cell that produces them uncleavedbetween about position 101 to about position 161 (including at the furincleavage sites at positions 106-109 and 131-136). If desired, theuncleaved RSV F protein ecto-domain polypeptide with altered furincleavage sites further contains altered or deleted sites for otherproteases (e.g., trypsin cleavage sites) between about position 101 andabout position 161 to prevent protease (e.g., trypsin) cleavage. Forexample, one or more lysine and/or arginine residues (e.g., all lysingand arginine residues) present between about position 101 and aboutposition 161 are deleted or replaced with an amino acid that is notlysine or arginine, and the RSV F protein ecto-domain polypeptides arenot cleaved by trypsin between about position 101 and about position161.

The uncleaved RSV F protein ecto-domain polypeptide monomers, trimersand combinations of monomers and trimers can be purified to the desireddegree. It is generally preferred that the uncleaved RSV F proteinecto-domain polypeptide monomers or trimers are purified, for example,to be at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 95% or substantially homogenous. Asdescribed herein, uncleaved RSV F protein ecto-domain polypeptides canbe readily purified from lipids and lipoproteins, for example, by sizeexclusion chromatography. Accordingly, the method can be used to readilyproduce a composition containing cleaved RSV F protein ecto-domainpolypeptide monomers, trimers, or a combination of monomers and trimersthat are substantially free of lipids and lipoproteins.

In one example, the method includes providing insect cell cultureconditioned medium, mammalian cell culture conditioned medium, aviancell conditioned medium, yeast cell conditioned medium, Tetrahymena cellconditioned medium, or a combination thereof. In some embodiments,uncleaved RSV F protein ecto-domain polypeptide trimers are purified. Inother embodiments, uncleaved RSV F protein ecto-domain polypeptidemonomers are purified. In other embodiments, uncleaved RSV F proteinecto-domain polypeptide monomers and trimers are purified.

Methods for Producing Cleaved RSV F Protein Ecto-Domain Polypeptideswith Altered Fusion Peptides

In one aspect, the invention is a method for preparing a compositionthat contains cleaved RSV F protein ecto-domain polypeptides thatcontain an altered fusion peptide. When RSV F protein ecto-domainpolypeptides that do not contain altered furin cleavage sites areexpressed in host cells, the host cells process the polypeptides, inpart by cleaving the polypeptide at the furin sites at about positions109/110 and about positions 136/137 to produce F₁ and F₂ subunits. Theprocessed polypeptides are secreted into the culture and can berecovered as associated F₁-F₂ subunits (e.g., disulphide bonding F₁ andF₂ subunits), which can form rosettes of trimers through aggregation ofexposed fusion peptides. RSV F protein ecto-domain polypeptides thatcontain altered fusion peptides can be produced in and secreted fromhost cells as associated F₁-F₂ subunits, and preferably do not aggregateinto rosettes or with lipids or lipoprotein contaminants. Withoutwishing to be bound by any particular theory, it is believed that thepolypeptides do not form rosettes or associate with lipid andlipoprotein contaminants because the altered fusion peptide does notmediate aggregation.

The method of this aspect of the invention includes: a) providing abiological material that contains cleaved RSV F protein ecto-domainpolypeptides that contain an altered fusion peptide (e.g., at least aportion of the fusion peptide is deleted), such as a cell lysate, cellhomogenate or cell culture conditioned medium; and b) purifying cleavedRSV F protein ecto-domain polypeptides from the biological material. Thepurified cleaved RSV F protein ecto-domain polypeptides can be purifiedas cleaved trimers, rosettes of cleaved trimers, or a mixture of cleavedtrimers and rosettes of cleaved trimers. Suitable RSV F proteinecto-domain polypeptides that contain altered fusion peptides containcleavable furin cleavage sites at about 109/110 and about 136/137 andfurther contain an altered fusion peptide as described herein. Forexample, an RSV F protein ecto-domain polypeptide in which about aminoacids 137-152 are deleted, about amino acids 137-153 are deleted, aboutamino acids 137-145 are deleted, about amino acids 137-146 are deletedor about amino acids 137-142 are deleted, can be used in the method. Inparticular examples, the biological material that contains uncleaved RSVF protein ecto-domain polypeptides; includes at least the fusion peptidedeletion 1.

The cleaved RSV F protein ecto-domain polypeptides (e.g., cleavedtrimers or a mixture of cleaved trimers and rosettes of cleaved trimers)can be purified to the desired degree. It is generally preferred thatthe cleaved RSV F protein ecto-domain polypeptides are purified, forexample, to be at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 95% or substantially homogenous.As described herein, cleaved RSV F protein ecto-domain polypeptides thatcontain an altered fusion peptide can be readily purified from lipidsand lipoproteins, for example, by size exclusion chromatography.Accordingly, the method can be used to readily produce a compositioncontaining cleaved RSV F protein ecto-domain polypeptide trimers,rosettes of cleaved trimers, or a combination of cleaved trimers androsettes of cleaved trimers that are substantially free of lipids andlipoproteins.

Methods for Producing RSV F Protein Ecto-Domain Polypeptides withC-Terminal Furin Mutations

In another aspect, the invention is a method for preparing a compositionthat contains C-terminal uncleaved RSV ecto-domain polypeptides and amethod for preparing cleaved RSV F protein ecto-domain polypeptides.Without wishing to be bound by any particular theory, it is believedthat C-terminal uncleaved RSV F protein ecto-domain polypeptides arecleaved by cells that produce the proteins at the furin cleavage site atabout position 109/110, but not at the furin cleavage site at aboutposition 136/137, and are secreted into the media as an F₁ subunit thatis associated with an F₂ subunit. It is further believed that thehydrophobic fusion peptide is not exposed in the C-terminal uncleavedRSV F protein ecto-domain polypeptides and, therefore, the C-terminaluncleaved polypeptides do not associate with lipid and lipoproteincontaminants. As further described herein, C-terminal uncleaved RSV Fprotein ecto-domains can be cleaved further to produce a F₁ subunit, inwhich the amino terminus is from position 110 to about position 161,that is associated with a F₂ subunit. Such F₁ and F₂ subunits, which canbe purified as trimers, rosettes of trimers, or a mixture of trimers androsettes of trimers.

Generally, the amino acid sequence of a C-terminal uncleaved RSV Fprotein ecto-domain is altered to prevent cleavage at the furin cleavagesite at about position 136/137, but contains a naturally occurring orintroduced protease cleavage site, that when cleaved produces a F₁subunit, in which the amino terminus is from position 110 to aboutposition 161, and a F₂ subunit. For example, the C-terminal uncleavedRSV F protein ecto-domain polypeptide can have an amino acid sequencethat is altered to prevent cleavage at the furin cleavage sites at aboutposition 136/137, but contain one or more naturally occurring orintroduced protease cleavage sites from about position 101 to aboutposition 161. In a particular example, the amino acid sequence of aC-terminal uncleaved RSV F protein ecto-domain is altered to preventcleavage at the furin cleavage site at about position 136/137, butcontains a naturally occurring furin cleavage site at about position109/110.

A variety of particular amino acid sequences that will allow C-terminaluncleaved RSV F protein ecto-domain polypeptides to be produced andexpressed by host cells, including amino acid sequences that are notcleaved at the furin cleavage sites at about position 136/137, can bereadily designed and envisioned by a person of ordinary skill in theart. In general, one or more amino acids that are part of, or arelocated near by, the furin cleavage sites at about position 136/137 areindependently replaced or deleted. Suitable amino acid substitutions anddeletions that prevent cleavage at about position 136/137 are describedherein. For example, the substitution K131Q, the deletion of the aminoacids at positions 131-134, or the substitutionsK131Q/R133Q/R135Q/R136Q, each of which inhibit cleavage at 136/137, canbe used. In certain embodiments, C-terminal uncleaved RSV F proteinecto-domain polypeptides comprise at least one amino acid substitutionor deletion at about positions 133-136.

Similarly, a variety of particular amino acid sequences of C-terminaluncleaved RSV F protein ecto-domain polypeptides that contain a proteasecleavage site (e.g., naturally occurring or introduced) that whencleaved produce a first subunit that comprises an F₁ and a secondsubunit that comprises F₂, are possible and can be readily designed andenvisioned. For example, the amino acid sequence of RSV F protein fromabout position 101 to about position 161 contains trypsin cleavagesites, and one or more of the trypsin cleavage sites can be cleaved bytrypsin to generate F₁ and F₂ subunits. If desired, one or more suitableprotease recognition sites can be introduced into the C-terminaluncleaved RSV F protein ecto-domain polypeptide, for example, betweenabout positions 101 to about position 161. The introduced proteaserecognition sites can be cleaved using the appropriate protease togenerate F₁ and F₂ subunits. When a protease recognition site isintroduced into the amino acid sequence of a C-terminal uncleaved RSV Fprotein ecto-domain polypeptide, it is preferred that the site isrecognized by a protease that does not cleave the ecto-domain ofnaturally occurring RSV F protein.

C-terminal uncleaved RSV F protein ecto-domain polypeptides can beproduced using any suitable method. A preferred method is by recombinantexpression of constructs that encode a RSV F protein ecto-domain inwhich that amino acid sequence of the furin cleavage site at aboutpositions 136/137 is altered, so that the C-terminal uncleaved RSV Fprotein ecto-domain polypeptides are secreted by a host cell thatproduces the polypeptides uncleaved at the furin cleavage site at aboutposition 136/137. Preferably, the C-terminal uncleaved RSV F proteinecto-domain polypeptide is secreted by a host cell that produces it asan F₁ subunit that is associated with an F₂ subunit, wherein the aminoterminus of the F₁ subunit is from position 132 to about position 161,but not position 137. The C-terminal uncleaved RSV F protein ecto-domainpolypeptides can be produced using any suitable host cell, as describedherein.

One method of this aspect of the invention includes: a) providingC-terminal uncleaved RSV F protein ecto-domain polypeptides thatcomprise an altered furin cleavage site at position 136/137, and saidC-terminal uncleaved RSV F protein ecto-domain polypeptides are secretedfrom a cell that produces them in the form of an F₂ fragment that isassociated with a subunit that comprises F₁ but is uncleaved at position136/137, and b) cleaving the provided C-terminal uncleaved RSV F proteinecto-domain polypeptides with a protease that cleaves RSV F proteinecto-domain at a site between positions 101 and 161, thereby producingsaid composition. In particular embodiments, step b) comprises cleavingthe provided C-terminal uncleaved RSV F protein ecto-domain polypeptideswith a protease that cleaves RSV F protein ecto-domain at a site betweenabout positions 101 and 132, or about positions 132 and 161, or aboutpositions 110 and 132. Alternatively or in addition, in someembodiments, the C-terminal uncleaved RSV F protein ecto-domainpolypeptides comprise an altered furin cleavage site at position136/137, with the proviso that the altered furin cleavage site is notdeletion of amino acids 131-134. In particular examples, the biologicalmaterial that contains C-terminal uncleaved RSV F protein ecto-domainpolypeptides; includes at least the N-term Furin polypeptide.

The provided C-terminal uncleaved RSV F protein ecto-domain polypeptidescan be purified to the desired degree. For example, the providedC-terminal uncleaved RSV F protein ecto-domain polypeptides can beprovided in a cell lysate, cell homogenate, or cell culture conditionedmedia that is substantially unprocessed (e.g., unprocessed, or clarifiedonly), or in partially or substantially purified form. In particularexamples, the provided C-terminal uncleaved RSV F protein ecto-domainpolypeptides are provided in cell culture conditioned media selectedfrom the group consisting of insect cell conditioned media, mammaliancell conditioned media, avian cell conditioned media, yeast cellconditioned media, Tetrahymena cell conditioned media, and combinationsthereof.

It is generally preferred that the provided C-terminal uncleaved RSV Fprotein ecto-domain polypeptides are purified, for example, purified tobe at least about 80%, at least about 85%, at least about 90%, at leastabout 95% or substantially homogenous. As described herein, C-terminaluncleaved RSV F protein ecto-domain polypeptides can be readily purifiedfrom lipids and lipoproteins, while conventionally produced cleavedforms of RSV F protein co-purify with lipid and lipoproteincontaminants. Accordingly, when purified C-terminal uncleaved RSV Fprotein ecto-domain polypeptides are provided, the method can be used toreadily produce a composition containing cleaved RSV F proteinecto-domains that are substantially free of lipids or phospholipids.

Suitable methods for cleaving polypeptides using a protease arewell-known and conventional in the art. Generally, the polypeptides tobe cleaved are combined with a sufficient amount of protease underconditions (e.g., pH, polypeptide and protease concentration,temperature) suitable for cleavage of the polypeptide. Many suitableproteases are commercially available, and suitable conditions forperforming polypeptide cleavage are well-known for many proteases. Ifdesired, the RSV F protein ecto-domain polypeptides can be purifiedfollowing cleavage with protease.

In one example of the method, C-terminal uncleaved RSV F proteinecto-domain polypeptides are provided that contain an intact fusionpeptide, such as a C-terminal uncleaved RSV F protein ecto-domainpolypeptide in which none of the amino acids from positions 137-154 aresubstituted or deleted. In another example of the method, C-terminaluncleaved RSV F protein ecto-domain polypeptides are provided thatcontain an altered fusion peptide, such as a C-terminal uncleaved RSV Fprotein ecto-domain polypeptide in which about amino 137-152, aboutamino acids 137-153, about amino acids 137-145 or about amino acids137-142 are deleted. Other suitable fusion peptide deletions have alsobeen described, such as the deletion of the amino acids at positions137-146. Ruiz-Arguello et al., J. Gen. Virol., 85:3677-3687 (2004).

In some embodiments, the provided C-terminal uncleaved RSV F proteinecto-domain polypeptides are purified. The provided uncleaved RSV Fprotein ecto-domain polypeptides are cleaved, and cleavage results inthe formation of trimers of cleaved RSV F protein ecto-domainpolypeptides. If desired, the trimers can be purified further using anysuitable methods, such as size exclusion chromatography.

In particular examples of the method, the provided C-terminal uncleavedRSV F protein ecto-domain polypeptides contain at least the N-terminalFurin polypeptide (FIGS. 1A-1C). The provided C-terminal uncleaved RSV Fprotein ecto-domain polypeptides are cleaved, for example with trypsin,and cleavage results in the formation of cleaved trimers, rosettes ofcleaved trimers, or a combination of cleaved trimers and rosettes ofcleaved trimers of RSV F protein ecto-domain polypeptides. If desired,the cleaved trimers and/or rosettes of cleaved trimers can be purifiedfurther using any suitable methods, such as size exclusionchromatography.

Another method of this aspect of the invention includes: a) providing abiological material, such as a cell lysate, cell homogenate or cellculture conditioned medium, that contains a C-terminal uncleaved RSV Fprotein ecto-domain polypeptides that comprise an altered furin cleavagesite at position 136/137, and said soluble RSV F protein ecto-domainpolypeptides are secreted from a cell that produces them in the form ofan F₂ fragment that is associated with a subunit that comprises F₁ butis uncleaved at position 136/137, with the proviso that the alteredfurin cleavage site is not deletion of amino acids 131-134; and b)purifying the C-terminal uncleaved RSV F protein ecto-domainpolypeptides from the biological material, thereby producing thecomposition. Preferably, the amino terminus of the F₁ subunit is fromabout position 110 to about position 132. More preferably, the aminoterminus of the F₁ subunit is about position 110. It is particularlypreferred that the amino terminus of the F₁ subunit is not position 137.In particular examples, the biological material that contains C-terminaluncleaved RSV F protein ecto-domain polypeptides; includes at least theN-term Furin polypeptide.

If desired, the C-terminal uncleaved RSV F protein ecto-domainpolypeptide further contains altered or deleted sites for otherproteases (e.g., trypsin cleavage sites) between about position 101 andabout position 161 to prevent protease (e.g., trypsin) cleavage. Forexample, one or more lysine and/or arginine residues (e.g., all lysineand arginine residues) present between about position 101 and aboutposition 161 are deleted or replaced with an amino acid that is notlysine or arginine, and the C-terminal uncleaved RSV F proteinecto-domain polypeptides are not cleaved by trypsin between aboutposition 101 and about position 161. The C-terminal uncleaved RSV Fprotein ecto-domain polypeptides can contain an intact fusion peptide oran altered fusion peptide, as described herein.

The C-terminal uncleaved RSV F protein ecto-domain polypeptides, e.g.,monomers, trimers and combinations of monomers and trimers can bepurified to the desired degree. It is generally preferred that theC-terminal uncleaved RSV F protein ecto-domain polypeptide monomers ortrimers are purified, for example, to be at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95% orsubstantially homogenous. As described herein, C-terminal uncleaved RSVF protein ecto-domain polypeptides can be readily purified from lipidsand lipoproteins, for example, by size exclusion chromatography.Accordingly, the method can be used to readily produce a compositioncontaining C-terminal uncleaved RSV F protein ecto-domain polypeptides,e.g., monomers, trimers, or a combination of monomers and trimers, thatare substantially free of lipids and lipoproteins. In particularexamples of the method, the C-terminal uncleaved RSV F proteinecto-domain polypeptides contain at least the N-terminal Furinpolypeptide (FIGS. 1A-1C).

In one example, the method includes providing insect cell cultureconditioned medium, mammalian cell culture conditioned medium, aviancell conditioned media, yeast cell conditioned media, Tetrahymena cellconditioned media or a combination thereof. In some embodiments,C-terminal uncleaved RSV F protean ecto-domain polypeptide trimers arepurified. In other embodiments, C-terminal uncleaved RSV F proteinecto-domain polypeptide monomers are purified. In other embodiments,C-terminal uncleaved RSV F protein ecto-domain polypeptide monomers andtrimers are purified.

Self-Replicating RNA

The RSV-F polypeptides described herein can be produced by expression ofrecombinant nucleic acids that encode the polypeptides in the cells of asubject. Preferred nucleic acids that can be administered to a subjectto cause the production of RSV-F polypeptides are self-replicating RNAmolecules. The self-replicating RNA molecules of the invention are basedon the genomic RNA of RNA viruses, but lack the genes encoding one ormore structural proteins. The self-replicating RNA molecules are capableof being translated to produce non-structural proteins of the RNA virusand heterologous proteins encoded by the self-replicating RNA.

The self-replicating RNA generally contains at least one or more genesselected from the group consisting of viral replicase, viral proteases,viral helicases and other nonstructural viral proteins, and alsocomprise 5′- and 3′-end cis-active replication sequences, and ifdesired, a heterologous sequences that encode a desired amino acidsequences (e.g., a protein, an antigen). A subgenomic promoter thatdirects expression of the heterologous sequence can be included in theself-replicating RNA. If desired, the heterologous sequence may be fusedin frame to other coding regions in the self-replicating RNA and/or maybe under the control of an internal ribosome entry site (IRES).

Self-replicating RNA molecules of the invention can be designed so thatthe self-replicating RNA molecule cannot induce production of infectiousviral particles. This can be achieved, for example, by omitting one ormore viral genes encoding structural proteins that are necessary for theproduction of viral particles in the self-replicating RNA. For example,when the self-replicating RNA molecule is based on an alpha virus, suchas Sinebis virus (SIN), Semliki forest virus and Venezuelan equineencephalitis virus (VEE), one or more genes encoding viral structuralproteins, such as capsid and/or envelope glycoproteins, can be omitted.If desired, self-replicating RNA molecules of the invention can bedesigned to induce production of infectious viral particles that areattenuated or virulent, or to produce viral particles that are capableof a single round of subsequent infection.

A self-replicating RNA molecule can, when delivered to a vertebrate celleven without any proteins, lead to the production of multiple daughterRNAs by transcription from itself (or from an antisense copy of itself).The self-replicating RNA can be directly translated after delivery to acell, and this translation provides a RNA-dependent RNA polymerase whichthen produces transcripts from the delivered RNA. Thus the delivered RNAleads to the production of multiple daughter RNAs. These transcripts areantisense relative to the delivered RNA and may be translated themselvesto provide in situ expression of a gene product, or may be transcribedto provide further transcripts with the same sense as the delivered RNAwhich are translated to provide in situ expression of the encoded RSV-Fpolypeptide.

One suitable system for achieving self-replication is to use analphavirus-based RNA replicon. These + stranded replicons are translatedafter delivery to a cell to give of a replicase (orreplicase-transcriptase). The replicase is translated as a polyproteinwhich auto cleaves to provide a replication complex which createsgenomic − strand copies of the + strand delivered RNA. These − strandtranscripts can themselves be transcribed to give further copies ofthe + stranded parent RNA and also to give a subgenomic transcript whichencodes the RSV-F polypeptide. Translation of the subgenomic transcriptthus leads to in situ expression of the RSV-F polypeptide by theinfected cell. Suitable alphavirus replicons can use a replicase from asindbis virus, a semliki forest virus, an eastern equine encephalitisvirus, a venezuelan equine encephalitis virus, etc.

A preferred self-replicating RNA molecule thus encodes (i) aRNA-dependent RNA polymerase which can transcribe RNA from theself-replicating RNA molecule and (ii) an RSV-F polypeptide. Thepolymerase can be an alphavirus replicase e.g. comprising alphavirusprotein nsP4.

Whereas natural alphavirus genomes encode structural virion proteins inaddition to the non structural replicase polyprotein, it is preferredthat an alphavirus based self-replicating RNA molecule of the inventiondoes not encode alphavirus structural proteins. Thus the selfreplicating RNA can lead to the production of genomic RNA copies ofitself in a cell, but not to the production of RNA-containing alphavirusvirions. The inability to produce these virions means that, unlike awild-type alphavirus, the self-replicating RNA molecule cannotperpetuate itself in infectious form. The alphavirus structural proteinswhich are necessary for perpetuation in wild-type viruses are absentfrom self replicating RNAs of the invention and their place is taken bygene(s) encoding the desired gene product, such that the subgenomictranscript encodes the desired gene product rather than the structuralalphavirus virion proteins.

Thus a self-replicating RNA molecule useful with the invention may havetwo open reading frames. The first (5′) open reading frame encodes areplicase; the second (3′) open reading frame encodes an RSV-Fpolypeptide. In some embodiments the RNA may have additional(downstream) open reading frames e.g. that encode further desired geneproducts. A self-replicating RNA molecule can have a 5′ sequence whichis compatible with the encoded replicase.

In one aspect, the self-replicating RNA molecule is derived from orbased on an alphavirus. In other aspects, the self-replicating RNAmolecule is derived from or based on a virus other than an alphavirus,preferably, a positive-stranded RNA viruses, and more preferably apicornavirus, flavivirus, rubivirus, pestivirus, hepacivirus,calicivirus, or coronavirus. Suitable wild-type alphavirus sequences arewell-known and are available from sequence depositories, such as theAmerican Type Culture Collection, Rockville, Md. Representative examplesof suitable alphaviruses include Aura (ATCC VR-368), Bebaru virus (ATCCVR-600, ATCC VR-1240), Cabassou (ATCC VR-922), Chikungunya virus (ATCCVR-64, ATCC VR-1241), Eastern equine encephalomyelitis virus (ATCCVR-65, ATCC VR-1242), Fort Morgan (ATCC VR-924), Getah virus (ATCCVR-369, ATCC VR-1243), Kyzylagach (ATCC VR-927), Mayaro (ATCC VR-66),Mayaro virus (ATCC VR-1277), Middleburg (ATCC VR-370), Mucambo virus(ATCC VR-580, ATCC VR-1244), Ndumu (ATCC VR-371), Pixuna virus (ATCCVR-372, ATCC VR-1245), Ross River virus (ATCC VR-373, ATCC VR-1246),Semliki Forest (ATCC VR-67, ATCC VR-1247), Sindbis virus (ATCC VR-68,ATCC VR-1248), Tonate (ATCC VR-925), Triniti (ATCC VR-469), Una (ATCCVR-374), Venezuelan equine encephalomyelitis (ATCC VR-69, ATCC VR-923,ATCC VR-1250 ATCC VR-1249, ATCC VR-532), Western equineencephalomyelitis (ATCC VR-70, ATCC VR-1251, ATCC VR-622, ATCC VR-1252),Whataroa (ATCC VR-926), and Y-62-33 (ATCC VR-375).

The self-replicating RNA may be associated with a delivery system. Theself-replicating RNA may be administered with or without an adjuvant.

RNA Delivery Systems

The self-replicating RNA of the invention are suitable for delivery in avariety of modalities, such as naked RNA delivery or in combination withlipids, polymers or other compounds that facilitate entry into thecells. Self-replicating RNA molecules of the present invention can beintroduced into target cells or subjects using any suitable technique,e.g., by direct injection, microinjection, electroporation, lipofection,biolystics, and the like. The self-replicating RNA molecule may also beintroduced into cells by way of receptor-mediated endocytosis. See e.g.,U.S. Pat. No. 6,090,619; Wu and Wu, J. Biol. Chem., 263:14621 (1988);and Curiel et al., Proc. Natl. Acad. Sci. USA, 88:8850 (1991). Forexample, U.S. Pat. No. 6,083,741 discloses introducing an exogenousnucleic acid into mammalian cells by associating the nucleic acid to apolycation moiety (e.g., poly-L-lysine having 3-100 lysine residues),which is itself coupled to an integrin receptor-binding moiety (e.g., acyclic peptide having the sequence Arg-Gly-Asp).

The self-replicating RNA molecule of the present invention can bedelivered into cells via amphiphiles. See e.g., U.S. Pat. No. 6,071,890.Typically, a nucleic acid molecule may form a complex with the cationicamphiphile. Mammalian cells contacted with the complex can readily takeit up.

The self-replicating RNA can be delivered as naked RNA (e.g. merely asan aqueous solution of RNA) but, to enhance entry into cells and alsosubsequent intercellular effects, the self-replicating RNA is preferablyadministered in combination with a delivery system, such as aparticulate or emulsion delivery system. A large number of deliverysystems are well known to those of skill in the art. Such deliverysystems include, for example liposome-based delivery (Debs and Zhu(1993) WO 93/24640; Mannino and Gould-Fogerite (1988) BioTechniques6(7): 682-691; Rose U.S. Pat. No. 5,279,833; Brigham (1991) WO 91/06309;and Felgner et al. (1987) Proc. Natl. Acad. Sci. USA 84: 7413-7414), aswell as use of viral vectors (e.g., adenoviral (see, e.g., Berns et al.(1995) Ann. NY Acad. Sci. 772: 95-104; Ali et al. (1994) Gene Ther. 1:367-384; and Haddada et al. (1995) Curr. Top. Microbiol. Immunol. 199(Pt 3): 297-306 for review), papillomaviral, retroviral (see, e.g.,Buchscher et al. (1992) J. Virol. 66(5) 2731-2739; Johann et al. (1992)J. Virol. 66 (5): 1635-1640 (1992); Sommerfelt et al., (1990) Virol.176:58-59; Wilson et al. (1989) J. Virol. 63:2374-2378; Miller et al.,J. Virol. 65:2220-2224 (1991); Wong-Staal et al., PCT/US94/05700, andRosenburg and Fauci (1993) in Fundamental Immunology, Third Edition Paul(ed) Raven Press, Ltd., New York and the references therein, and Yu etal., Gene Therapy (1994) supra.), and adeno-associated viral vectors(see, West et al. (1987) Virology 160:38-47; Carter et al. (1989) U.S.Pat. No. 4,797,368; Carter et al. WO 93/24641 (1993); Kotin (1994) HumanGene Therapy 5:793-801; Muzyczka (1994) J. Clin. Invst. 94:1351 andSamulski (supra) for an overview of AAV vectors; see also, Lebkowski,U.S. Pat. No. 5,173,414; Tratschin et al. (1985) Mol. Cell. Biol.5(11):3251-3260; Tratschin, et al. (1984) Mol. Cell. Biol., 4:2072-2081;Hermonat and Muzyczka (1984) Proc. Natl. Acad. Sci. USA, 81:6466-6470;McLaughlin et al. (1988) and Samulski et al. (1989) J. Virol.,63:03822-3828), and the like.

Three particularly useful delivery systems are (i) liposomes (ii)non-toxic and biodegradable polymer microparticles (iii) cationicsubmicron oil-in-water emulsions.

Liposomes

Various amphiphilic lipids can form bilayers in an aqueous environmentto encapsulate a RNA-containing aqueous core as a liposome. These lipidscan have an anionic, cationic or zwitterionic hydrophilic head group.Formation of liposomes from anionic phospholipids dates back to the1960s, and cationic liposome-forming lipids have been studied since the1990s. Some phospholipids are anionic whereas other are zwitterionic.Suitable classes of phospholipid include, but are not limited to,phosphatidylethanolamines, phosphatidylcholines, phosphatidylserines,and phosphatidylglycerols, and some useful phospholipids are listed inTable 20. Useful cationic lipids include, but are not limited to,dioleoyl trimethylammonium propane (DOTAP),1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA),1,2-dioleyloxy-N,Ndimethyl-3-aminopropane (DODMA),1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA),1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane (DLenDMA). Zwitterioniclipids include, but are not limited to, acyl zwitterionic lipids andether zwitterionic lipids. Examples of useful zwitterionic lipids areDPPC, DOPC and dodecylphosphocholine. The lipids can be saturated orunsaturated.

Liposomes can be formed from a single lipid or from a mixture of lipids.A mixture may comprise (i) a mixture of anionic lipids (ii) a mixture ofcationic lipids (iii) a mixture of zwitterionic lipids (iv) a mixture ofanionic lipids and cationic lipids (v) a mixture of anionic lipids andzwitterionic lipids (vi) a mixture of zwitterionic lipids and cationiclipids or (vii) a mixture of anionic lipids, cationic lipids andzwitterionic lipids. Similarly, a mixture may comprise both saturatedand unsaturated lipids. For example, a mixture may comprise DSPC(zwitterionic, saturated), DlinDMA (cationic, unsaturated), and/or DMPG(anionic, saturated). Where a mixture of lipids is used, not all of thecomponent lipids in the mixture need to be amphiphilic e.g. one or moreamphiphilic lipids can be mixed with cholesterol.

The hydrophilic portion of a lipid can be PEGylated (i.e. modified bycovalent attachment of a polyethylene glycol). This modification canincrease stability and prevent non-specific adsorption of the liposomes.For instance, lipids can be conjugated to PEG using techniques such asthose disclosed in Heyes et al. (2005) J Controlled Release 107:276-287.

A mixture of DSPC, DlinDMA, PEG-DMPG and cholesterol is used in theexamples. A separate aspect of the invention is a liposome comprisingDSPC, DlinDMA, PEG-DMG and cholesterol. This liposome preferablyencapsulates RNA, such as a self-replicating RNA e.g. encoding animmunogen.

Liposomes are usually divided into three groups: multilamellar vesicles(MLV); small unilamellar vesicles (SUV); and large unilamellar vesicles(LUV). MLVs have multiple bilayers in each vesicle, forming severalseparate aqueous compartments. SUVs and LUVs have a single bilayerencapsulating an aqueous core; SUVs typically have a diameter ≤50 nm,and LUVs have a diameter >50 nm. Liposomes useful with of the inventionare ideally LUVs with a diameter in the range of 50-220 nm. For acomposition comprising a population of LUVs with different diameters:(i) at least 80% by number should have diameters in the range of 20-220nm, (ii) the average diameter (Zav, by intensity) of the population isideally in the range of 40-200 nm, and/or (iii) the diameters shouldhave a polydispersity index <0.2.

Techniques for preparing suitable liposomes are well known in the arte.g. see Liposomes: Methods and Protocols, Volume 1: PharmaceuticalNanocarriers: Methods and Protocols. (ed. Weissig). Humana Press, 2009.ISBN 160327359X; Liposome Technology, volumes I, II & III. (ed.Gregoriadis). Informa Healthcare, 2006; and Functional Polymer Colloidsand Microparticles volume 4 (Microspheres, microcapsules & liposomes).(eds. Arshady & Guyot). Citus Books, 2002. One useful method involvesmixing (i) an ethanolic solution of the lipids (ii) an aqueous solutionof the nucleic acid and (iii) buffer, followed by mixing, equilibration,dilution and purification (Heyes et al. (2005) J Controlled Release107:276-87.).

RNA is preferably encapsulated within the liposomes, and so the liposomeforms a outer layer around an aqueous RNA-containing core. Thisencapsulation has been found to protect RNA from RNase digestion. Theliposomes can include some external RNA (e.g. on the surface of theliposomes), but at least half of the RNA (and ideally all of it) isencapsulated.

Polymeric Microparticles

Various polymers can form microparticles to encapsulate or adsorb RNA.The use of a substantially non-toxic polymer means that a recipient cansafely receive the particles, and the use of a biodegradable polymermeans that the particles can be metabolised after delivery to avoidlong-term persistence. Useful polymers are also sterilisable, to assistin preparing pharmaceutical grade formulations.

Suitable non-toxic and biodegradable polymers include, but are notlimited to, poly(α-hydroxy acids), polyhydroxy butyric acids,polylactones (including polycaprolactones), polydioxanones,polyvalerolactone, polyorthoesters, polyanhydrides, polycyanoacrylates,tyrosine-derived polycarbonates, polyvinyl-pyrrolidinones orpolyester-amides, and combinations thereof.

In some embodiments, the microparticles are formed from poly(α-hydroxyacids), such as a poly(lactides) (“PLA”), copolymers of lactide andglycolide such as a poly(D,L-lactide-co-glycolide) (“PLG”), andcopolymers of D,L-lactide and caprolactone. Useful PLG polymers includethose having a lactide/glycolide molar ratio ranging, for example, from20:80 to 80:20 e.g. 25:75, 40:60, 45:55, 55:45, 60:40, 75:25. Useful PLGpolymers include those having a molecular weight between, for example,5,000-200,000 Da e.g. between 10,000-100,000, 20,000-70,000,40,000-50,000 Da.

The microparticles ideally have a diameter in the range of 0.02 μm to 8μm. For a composition comprising a population of microparticles withdifferent diameters at least 80% by number should have diameters in therange of 0.03-7 μm.

Techniques for preparing suitable microparticles are well known in theart e.g. see Functional Polymer Colloids and Microparticles volume 4(Microspheres, microcapsules & liposomes). (eds. Arshady & Guyot). CitusBooks, 2002; Polymers in Drug Delivery. (eds. Uchegbu & Schatzlein). CRCPress, 2006. (in particular chapter 7) and Microparticulate Systems forthe Delivery of Proteins and Vaccines. (eds. Cohen & Bernstein). CRCPress, 1996. To facilitate adsorption of RNA, a microparticle mayinclude a cationic surfactant and/or lipid e.g. as disclosed in O'Haganet al. (2001) J Virology 75:9037-9043; and Singh et al. (2003)Pharmaceutical Research 20: 247-251. An alternative way of makingpolymeric microparticles is by molding and curing e.g. as disclosed inWO2009/132206.

Microparticles of the invention can have a zeta potential of between40-100 mV.

RNA can be adsorbed to the microparticles, and adsorption is facilitatedby including cationic materials (e.g. cationic lipids) in themicroparticle.

Oil-In-Water Cationic Emulsions

Oil-in-water emulsions are known for adjuvanting influenza vaccines e.g.the MF59™ adjuvant in the FLUAD™ product, and the AS03 adjuvant in thePREPANDRIX™ product. RNA delivery according to the present invention canutilise an oil-in-water emulsion, provided that the emulsion includesone or more cationic molecules. For instance, a cationic lipid can beincluded in the emulsion to provide a positive droplet surface to whichnegatively-charged RNA can attach.

The emulsion comprises one or more oils. Suitable oil(s) include thosefrom, for example, an animal (such as fish) or a vegetable source. Theoil is ideally biodegradable (metabolisable) and biocompatible. Sourcesfor vegetable oils include nuts, seeds and grains. Peanut oil, soybeanoil, coconut oil, and olive oil, the most commonly available, exemplifythe nut oils. Jojoba oil can be used e.g. obtained from the jojoba bean.Seed oils include safflower oil, cottonseed oil, sunflower seed oil,sesame seed oil and the like. In the grain group, corn oil is the mostreadily available, but the oil of other cereal grains such as wheat,oats, rye, rice, teff, triticale and the like may also be used. 6-10carbon fatty acid esters of glycerol and 1,2-propanediol, while notoccurring naturally in seed oils, may be prepared by hydrolysis,separation and esterification of the appropriate materials starting fromthe nut and seed oils. Fats and oils from mammalian milk aremetabolizable and so may be used. The procedures for separation,purification, saponification and other means necessary for obtainingpure oils from animal sources are well known in the art.

Most fish contain metabolizable oils which may be readily recovered. Forexample, cod liver oil, shark liver oils, and whale oil such asspermaceti exemplify several of the fish oils which may be used herein.A number of branched chain oils are synthesized biochemically in5-carbon isoprene units and are generally referred to as terpenoids.Squalane, the saturated analog to squalene, can also be used. Fish oils,including squalene and squalane, are readily available from commercialsources or may be obtained by methods known in the art.

Other useful oils are the tocopherols, particularly in combination withsqualene. Where the oil phase of an emulsion includes a tocopherol, anyof the α, β, γ, δ, ε or ξ tocopherols can be used, but α-tocopherols arepreferred. D-α-tocopherol and DL-α-tocopherol can both be used. Apreferred α-tocopherol is DL-α-tocopherol. An oil combination comprisingsqualene and a tocopherol (e.g. DL-α-tocopherol) can be used.

Preferred emulsions comprise squalene, a shark liver oil which is abranched, unsaturated terpenoid (C₃₀H₅₀;[(CH₃)₂C[═CHCH₂CH₂C(CH₃)]₂═CHCH₂—]₂;2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexaene; CAS RN7683-64-9).

The oil in the emulsion may comprise a combination of oils e.g. squaleneand at least one further oil.

The aqueous component of the emulsion can be plain water (e.g. w.f.i.)or can include further components e.g. solutes. For instance, it mayinclude salts to form a buffer e.g. citrate or phosphate salts, such assodium salts. Typical buffers include: a phosphate buffer; a Trisbuffer; a borate buffer; a succinate buffer; a histidine buffer; or acitrate buffer. A buffered aqueous phase is preferred, and buffers willtypically be included in the 5-20 mM range.

The emulsion also includes a cationic lipid. Preferably this lipid is asurfactant so that it can facilitate formation and stabilisation of theemulsion. Useful cationic lipids generally contains a nitrogen atom thatis positively charged under physiological conditions e.g. as a tertiaryor quaternary amine. This nitrogen can be in the hydrophilic head groupof an amphiphilic surfactant. Useful cationic lipids include, but arenot limited to: 1,2-dioleoyloxy-3-(trimethylammonio)propane (DOTAP),3′-[N—(N′,N′-Dimethylaminoethane)-carbamoyl]Cholesterol (DCCholesterol), dimethyldioctadecyl-ammonium (DDA e.g. the bromide),1,2-Dimyristoyl-3-Trimethyl-AmmoniumPropane (DMTAP),dipalmitoyl(C16:0)trimethyl ammonium propane (DPTAP),distearoyltrimethylammonium propane (DSTAP). Other useful cationiclipids are: benzalkonium chloride (BAK), benzethonium chloride,cetramide (which contains tetradecyltrimethylammonium bromide andpossibly small amounts of dedecyltrimethylammonium bromide andhexadecyltrimethyl ammonium bromide), cetylpyridinium chloride (CPC),cetyl trimethylammonium chloride (CTAC), N,N′,N′-polyoxyethylene(10)-N-tallow-1,3-diaminopropane, dodecyltrimethylammonium bromide,hexadecyltrimethyl-ammonium bromide, mixed alkyl-trimethyl-ammoniumbromide, benzyldimethyldodecylammonium chloride,benzyldimethylhexadecyl-ammonium chloride, benzyltrimethylammoniummethoxide, cetyldimethylethylammonium bromide, dimethyldioctadecylammonium bromide (DDAB), methylbenzethonium chloride, decamethoniumchloride, methyl mixed trialkyl ammonium chloride, methyltrioctylammonium chloride), N,N-dimethyl-N-[2(2-methyl-4-(1,1,3,3tetramethylbutyl)-phenoxy]-ethoxy)ethyl]-benzenemetha-naminiumchloride (DEBDA), dialkyldimetylammonium salts,[1-(2,3-dioleyloxy)-propyl]-N,N,N,trimethylammonium chloride,1,2-diacyl-3-(trimethylammonio) propane (acyl group=dimyristoyl,dipalmitoyl, distearoyl, dioleoyl), 1,2-diacyl-3(dimethylammonio)propane (acyl group=dimyristoyl, dipalmitoyl,distearoyl, dioleoyl),1,2-dioleoyl-3-(4′-trimethyl-ammonio)butanoyl-sn-glycerol, 1,2-dioleoyl3-succinyl-sn-glycerol choline ester, cholesteryl (4′-trimethylammonio)butanoate), N-alkyl pyridinium salts (e.g. cetylpyridinium bromide andcetylpyridinium chloride), N-alkylpiperidinium salts, dicationicbolaform electrolytes (C12Me6; C12BU6),dialkylglycetylphosphorylcholine, lysolecithin, L-αdioleoylphosphatidylethanolamine, cholesterol hemisuccinate cholineester, lipopolyamines, including but not limited todioctadecylamidoglycylspermine (DOGS), dipalmitoylphosphatidylethanol-amidospermine (DPPES), lipopoly-L (or D)-lysine(LPLL, LPDL), poly (L (or D)-lysine conjugated toN-glutarylphosphatidylethanolamine, didodecyl glutamate ester withpendant amino group (C{circumflex over ( )}GluPhCnN), ditetradecylglutamate ester with pendant amino group (Cl4GluCnN+), cationicderivatives of cholesterol, including but not limited tocholesteryl-3β-oxysuccinamidoethylenetrimethylammonium salt,cholesteryl-3β-oxysuccinamidoethylene-dimethylamine,cholesteryl-3β-carboxyamidoethylenetrimethylammonium salt, andcholesteryl-3β-carboxyamidoethylenedimethylamine. Other useful cationiclipids are described in US 2008/0085870 and US 2008/0057080, which areincorporated herein by reference.

The cationic lipid is preferably biodegradable (metabolisable) andbiocompatible.

In addition to the oil and cationic lipid, an emulsion can include anon-ionic surfactant and/or a zwitterionic surfactant. Such surfactantsinclude, but are not limited to: the polyoxyethylene sorbitan esterssurfactants (commonly referred to as the Tweens), especially polysorbate20 and polysorbate 80; copolymers of ethylene oxide (EO), propyleneoxide (PO), and/or butylene oxide (BO), sold under the DOWFAX™tradename, such as linear EO/PO block copolymers; octoxynols, which canvary in the number of repeating ethoxy (oxy-1,2-ethanediyl) groups, withoctoxynol-9 (Triton X-100, or t-octylphenoxypolyethoxyethanol) being ofparticular interest; (octylphenoxy)polyethoxyethanol (IGEPALCA-630/NP-40); phospholipids such as phosphatidylcholine (lecithin);polyoxyethylene fatty ethers derived from lauryl, cetyl, stearyl andoleyl alcohols (known as Brij surfactants), such as triethyleneglycolmonolauryl ether (Brij 30); polyoxyethylene-9-lauryl ether; and sorbitanesters (commonly known as the Spans), such as sorbitan trioleate (Span85) and sorbitan monolaurate. Preferred surfactants for including in theemulsion are polysorbate 80 (Tween 80; polyoxyethylene sorbitanmonooleate), Span 85 (sorbitan trioleate), lecithin and Triton X-100.

Mixtures of these surfactants can be included in the emulsion e.g. Tween80/Span 85 mixtures, or Tween 80/Triton-X100 mixtures. A combination ofa polyoxyethylene sorbitan ester such as polyoxyethylene sorbitanmonooleate (Tween 80) and an octoxynol such ast-octylphenoxy-polyethoxyethanol (Triton X-100) is also suitable.Another useful combination comprises laureth 9 plus a polyoxyethylenesorbitan ester and/or an octoxynol. Useful mixtures can comprise asurfactant with a HLB value in the range of 10-20 (e.g. polysorbate 80,with a HLB of 15.0) and a surfactant with a HLB value in the range of1-10 (e.g. sorbitan trioleate, with a HLB of 1.8).

Preferred amounts of oil (% by volume) in the final emulsion are between2-20% e.g. 5-15%, ⁶-14%, 7-13%, 8-12%. A squalene content of about 4-6%or about 9-11% is particularly useful.

Preferred amounts of surfactants (% by weight) in the final emulsion arebetween 0.001% and 8%. For example: polyoxyethylene sorbitan esters(such as polysorbate 80) 0.2 to 4%, in particular between 0.4-0.6%,between 0.45-0.55%, about 0.5% or between 1.5-2%, between 1.8-2.2%,between 1.9-2.10%, about 2%, or 0.85-0.95%, or about 10%; sorbitanesters (such as sorbitan trioleate) 0.02 to 2%, in particular about 0.5%or about 1%; octyl- or nonylphenoxy polyoxyethanols (such as TritonX-100) 0.001 to 0.1%, in particular 0.005 to 0.02%; polyoxyethyleneethers (such as laureth 9) 0.1 to 8%, preferably 0.1 to 10% and inparticular 0.1 to 1% or about 0.5%.

The absolute amounts of oil and surfactant, and their ratio, can bevaried within wide limits while still forming an emulsion. A skilledperson can easily vary the relative proportions of the components toobtain a desired emulsion, but a weight ratio of between 4:1 and 5:1 foroil and surfactant is typical (excess oil).

An important parameter for ensuring immunostimulatory activity of anemulsion, particularly in large animals, is the oil droplet size(diameter). The most effective emulsions have a droplet size in thesubmicron range. Suitably the droplet sizes will be in the range 50-750nm. Most usefully the average droplet size is less than 250 nm e.g. lessthan 200 nm, less than 150 nm. The average droplet size is usefully inthe range of 80-180 nm. Ideally, at least 80% (by number) of theemulsion's oil droplets are less than 250 nm in diameter, and preferablyat least 90%. Apparatuses for determining the average droplet size in anemulsion, and the size distribution, are commercially available. Thesetypically use the techniques of dynamic light scattering and/orsingle-particle optical sensing e.g. the Accusizer™ and Nicomp™ seriesof instruments available from Particle Sizing Systems (Santa Barbara,USA), or the Zetasizer™ instruments from Malvern Instruments (UK), orthe Particle Size Distribution Analyzer instruments from Horiba (Kyoto,Japan).

Ideally, the distribution of droplet sizes (by number) has only onemaximum i.e. there is a single population of droplets distributed aroundan average (mode), rather than having two maxima. Preferred emulsionshave a polydispersity of <0.4 e.g. 0.3, 0.2, or less.

Suitable emulsions with submicron droplets and a narrow sizedistribution can be obtained by the use of microfluidisation. Thistechnique reduces average oil droplet size by propelling streams ofinput components through geometrically fixed channels at high pressureand high velocity. These streams contact channel walls, chamber wallsand each other. The results shear, impact and cavitation forces cause areduction in droplet size. Repeated steps of microfluidisation can beperformed until an emulsion with a desired droplet size average anddistribution are achieved.

As an alternative to microfluidisation, thermal methods can be used tocause phase inversion. These methods can also provide a submicronemulsion with a tight particle size distribution.

Preferred emulsions can be filter sterilised i.e. their droplets canpass through a 220 nm filter. As well as providing a sterilisation, thisprocedure also removes any large droplets in the emulsion.

In certain embodiments, the cationic lipid in the emulsion is DOTAP. Thecationic oil-in-water emulsion may comprise from about 0.5 mg/ml toabout 25 mg/ml DOTAP. For example, the cationic oil-in-water emulsionmay comprise DOTAP at from about 0.5 mg/ml to about 25 mg/ml, from about0.6 mg/ml to about 25 mg/ml, from about 0.7 mg/ml to about 25 mg/ml,from about 0.8 mg/ml to about 25 mg/ml, from about 0.9 mg/ml to about 25mg/ml, from about 1.0 mg/ml to about 25 mg/ml, from about 1.1 mg/ml toabout 25 mg/ml, from about 1.2 mg/ml to about 25 mg/ml, from about 1.3mg/ml to about 25 mg/ml, from about 1.4 mg/ml to about 25 mg/ml, fromabout 1.5 mg/ml to about 25 mg/ml, from about 1.6 mg/ml to about 25mg/ml, from about 1.7 mg/ml to about 25 mg/ml, from about 0.5 mg/ml toabout 24 mg/ml, from about 0.5 mg/ml to about 22 mg/ml, from about 0.5mg/ml to about 20 mg/ml, from about 0.5 mg/ml to about 18 mg/ml, fromabout 0.5 mg/ml to about 15 mg/ml, from about 0.5 mg/ml to about 12mg/ml, from about 0.5 mg/ml to about 10 mg/ml, from about 0.5 mg/ml toabout 5 mg/ml, from about 0.5 mg/ml to about 2 mg/ml, from about 0.5mg/ml to about 1.9 mg/ml, from about 0.5 mg/ml to about 1.8 mg/ml, fromabout 0.5 mg/ml to about 1.7 mg/ml, from about 0.5 mg/ml to about 1.6mg/ml, from about 0.6 mg/ml to about 1.6 mg/ml, from about 0.7 mg/ml toabout 1.6 mg/ml, from about 0.8 mg/ml to about 1.6 mg/ml, about 0.5mg/ml, about 0.6 mg/ml, about 0.7 mg/ml, about 0.8 mg/ml, about 0.9mg/ml, about 1.0 mg/ml, about 1.1 mg/ml, about 1.2 mg/ml, about 1.3mg/ml, about 1.4 mg/ml, about 1.5 mg/ml, about 1.6 mg/ml, about 12mg/ml, about 18 mg/ml, about 20 mg/ml, about 21.8 mg/ml, about 24 mg/ml,etc. In an exemplary embodiment, the cationic oil-in-water emulsioncomprises from about 0.8 mg/ml to about 1.6 mg/ml DOTAP, such as 0.8mg/ml, 1.2 mg/ml, 1.4 mg/ml or 1.6 mg/ml.

In certain embodiments, the cationic lipid is DC Cholesterol. Thecationic oil-in-water emulsion may comprise DC Cholesterol at from about0.1 mg/ml to about 5 mg/ml DC Cholesterol. For example, the cationicoil-in-water emulsion may comprise DC Cholesterol from about 0.1 mg/mlto about 5 mg/ml, from about 0.2 mg/ml to about 5 mg/ml, from about 0.3mg/ml to about 5 mg/ml, from about 0.4 mg/ml to about 5 mg/ml, fromabout 0.5 mg/ml to about 5 mg/ml, from about 0.62 mg/ml to about 5mg/ml, from about 1 mg/ml to about 5 mg/ml, from about 1.5 mg/ml toabout 5 mg/ml, from about 2 mg/ml to about 5 mg/ml, from about 2.46mg/ml to about 5 mg/ml, from about 3 mg/ml to about 5 mg/ml, from about3.5 mg/ml to about 5 mg/ml, from about 4 mg/ml to about 5 mg/ml, fromabout 4.5 mg/ml to about 5 mg/ml, from about 0.1 mg/ml to about 4.92mg/ml, from about 0.1 mg/ml to about 4.5 mg/ml, from about 0.1 mg/ml toabout 4 mg/ml, from about 0.1 mg/ml to about 3.5 mg/ml, from about 0.1mg/ml to about 3 mg/ml, from about 0.1 mg/ml to about 2.46 mg/ml, fromabout 0.1 mg/ml to about 2 mg/ml, from about 0.1 mg/ml to about 1.5mg/ml, from about 0.1 mg/ml to about 1 mg/ml, from about 0.1 mg/ml toabout 0.62 mg/ml, about 0.15 mg/ml, about 0.3 mg/ml, about 0.6 mg/ml,about 0.62 mg/ml, about 0.9 mg/ml, about 1.2 mg/ml, about 2.46 mg/ml,about 4.92 mg/ml, etc. In an exemplary embodiment, the cationicoil-in-water emulsion comprises from about 0.62 mg/ml to about 4.92mg/ml DC Cholesterol, such as 2.46 mg/ml.

In certain embodiments, the cationic lipid is DDA. The cationicoil-in-water emulsion may comprise from about 0.1 mg/ml to about 5 mg/mlDDA. For example, the cationic oil-in-water emulsion may comprise DDA atfrom about 0.1 mg/ml to about 5 mg/ml, from about 0.1 mg/ml to about 4.5mg/ml, from about 0.1 mg/ml to about 4 mg/ml, from about 0.1 mg/ml toabout 3.5 mg/ml, from about 0.1 mg/ml to about 3 mg/ml, from about 0.1mg/ml to about 2.5 mg/ml, from about 0.1 mg/ml to about 2 mg/ml, fromabout 0.1 mg/ml to about 1.5 mg/ml, from about 0.1 mg/ml to about 1.45mg/ml, from about 0.2 mg/ml to about 5 mg/ml, from about 0.3 mg/ml toabout 5 mg/ml, from about 0.4 mg/ml to about 5 mg/ml, from about 0.5mg/ml to about 5 mg/ml, from about 0.6 mg/ml to about 5 mg/ml, fromabout 0.73 mg/ml to about 5 mg/ml, from about 0.8 mg/ml to about 5mg/ml, from about 0.9 mg/ml to about 5 mg/ml, from about 1.0 mg/ml toabout 5 mg/ml, from about 1.2 mg/ml to about 5 mg/ml, from about 1.45mg/ml to about 5 mg/ml, from about 2 mg/ml to about 5 mg/ml, from about2.5 mg/ml to about 5 mg/ml, from about 3 mg/ml to about 5 mg/ml, fromabout 3.5 mg/ml to about 5 mg/ml, from about 4 mg/ml to about 5 mg/ml,from about 4.5 mg/ml to about 5 mg/ml, about 1.2 mg/ml, about 1.45mg/ml, etc. Alternatively, the cationic oil-in-water emulsion maycomprise DDA at about 20 mg/ml, about 21 mg/ml, about 21.5 mg/ml, about21.6 mg/ml, about 25 mg/ml. In an exemplary embodiment, the cationicoil-in-water emulsion comprises from about 0.73 mg/ml to about 1.45mg/ml DDA, such as 1.45 mg/ml.

Catheters or like devices may be used to deliver the self-replicatingRNA molecules of the invention, as naked RNA or in combination with adelivery system, into a target organ or tissue. Suitable catheters aredisclosed in, e.g., U.S. Pat. Nos. 4,186,745; 5,397,307; 5,547,472;5,674,192; and 6,129,705, all of which are incorporated herein byreference.

The present invention includes the use of suitable delivery systems,such as liposomes, polymer microparticles or submicron emulsionmicroparticles with encapsulated or adsorbed self-replicating RNA, todeliver a self-replicating RNA molecule that encodes an RSV-Fpolypeptide, for example, to elicit an immune response alone, or incombination with another macromolecule. The invention includesliposomes, microparticles and submicron emulsions with adsorbed and/orencapsulated self-replicating RNA molecules, and combinations thereof.

As demonstrated further in the Examples, the self-replicating RNAmolecules associated with liposomes and submicron emulsionmicroparticles can be effectively delivered to the host cell, and caninduce an immune response to the protein encoded by the self-replicatingRNA.

The Immunogenic Composition

The invention provides immunogenic compositions. The immunogeniccompositions may include a single active immunogenic agent, or severalimmunogenic agents. For example, the immunogenic composition cancomprise RSV F polypeptides that are in a single form (e.g., monomer,trimer, or rosettes) or in two or more forms (e.g., a mixture of monomerand trimer or a dynamic equilibrium between monomer and trimer). Theimmunogenic composition can comprise a self-replicating RNA encoding anRSV-F polypeptide, and preferably also comprises a suitable deliverysystem, such as liposomes, polymeric microparticles, an oil-in-wateremulsion and combinations thereof.

Immunogenic compositions of the invention may also comprise one or moreimmunoregulatory agents. Preferably, one or more of the immunoregulatoryagents include one or more adjuvants, for example two, three, four ormore adjuvants. The adjuvants may include a TH1 adjuvant and/or a TH2adjuvant, further discussed below.

In another embodiment, an immunogenic composition of the inventioncomprises a polypeptide that displays an epitope present in a pre-fusionor an intermediate conformation of RSV-F glycoprotein, but does notdisplay the glycoprotein's post-fusion conformation.

In another embodiment, an immunogenic composition of the inventioncomprises a first polypeptide and a second polypeptide, wherein thefirst polypeptide comprises an RSV F protein, in whole or in part, andthe second polypeptide comprises a heterologous oligomerization domain.The first polypeptide can comprise an RSV F protein ectodomain. Thesecond polypeptide can be a trimerization domain from influenzahemagglutinin, a trimerization domain from SARS spike, a trimerizationdomain from HIV gp41, NadA, modified GCN4, or ATCase.

In one aspect, the invention is a composition comprising cleaved RSV Fprotein ecto-domain polypeptides produced by providing uncleaved RSV Fprotein ecto-domain polypeptides, or C-terminal uncleaved RSV F proteinecto-domain polypeptides, and cleaving them to produce F₁ and F₂subunits, as described herein.

In another aspect, the invention is a composition comprising uncleavedRSV F protein ecto-domain polypeptide trimers and/or monomers producedby providing a biological material that contains uncleaved RSV F proteinecto-domain polypeptides, and purifying uncleaved RSV F proteinecto-domain polypeptides monomers, uncleaved trimers, or a combinationof uncleaved monomers and uncleaved trimers (e.g., a mixture or adynamic equilibrium) from the biological material, as described herein.In some embodiments, the RSV F protein ecto-domain polypeptide containsaltered furin cleavage sites at about positions 106-109 and at aboutpositions 133-136, and if desired can further contain an altered fusionpeptide. In other embodiments, the RSV F protein ecto-domain containsaltered furin cleavage sites about positions 106-109 and at aboutpositions 133-136, and altered trypsin cleavage sites between aboutposition 101 and about position 161, and if desired can further containan altered fusion peptide.

In another aspect, the invention is a composition comprising C-terminaluncleaved RSV F protein ecto-domain polypeptide trimers and/or monomersproduced by providing a biological material that contains C-terminaluncleaved RSV F protein ecto-domain polypeptides, and purifyinguncleaved RSV F protein ecto-domain polypeptides monomers, uncleavedtrimers, or a combination of uncleaved monomers and uncleaved trimers(e.g., a mixture or a dynamic equilibrium) from the biological material,as described herein.

In another aspect, the invention is a composition comprising cleaved RSVF protein ecto-domain polypeptides produced by providing a biologicalmaterial that contains cleaved RSV F protein ecto-domain polypeptidesthat contain an altered fusion peptide (e.g., at least a portion of thefusion peptide is deleted) and purifying cleaved RSV F proteinecto-domain polypeptide trimers from the biological material, asdescribed herein.

In another aspect, the invention is a composition comprising uncleavedRSV F protein ecto-domain polypeptides produced by providing abiological material that contains uncleaved RSV F protein ecto-domainpolypeptides that contain an altered fusion peptide (e.g., at least aportion of the fusion peptide is deleted) and purifying uncleaved RSV Fprotein ecto-domain polypeptide monomers from the biological material,as described herein.

The compositions of the invention are preferably suitable foradministration to a mammalian subject, such as a human, and include oneor more pharmaceutically acceptable carrier(s) and/or excipient(s),including adjuvants. A thorough discussion of such components isavailable in reference 29. Compositions will generally be in aqueousform. When the composition is an immunogenic composition, it will elicitan immune response when administered to a mammal, such as a human. Theimmunogenic composition can be used to prepare a vaccine formulation forimmunizing a mammal.

The immunogenic compositions may include a single active immunogenicagent, or several immunogenic agents. For example, the RSV F proteinecto-domain polypeptide can be in a single form (e.g., uncleavedmonomer, cleaved monomer, uncleaved trimer, cleaved trimer, or rosettesof cleaved trimers) or in two or more forms (e.g., a mixture ofuncleaved monomer and uncleaved trimer or a dynamic equilibrium betweenuncleaved monomer and uncleaved trimer). In addition, the compositionscan contain an RSV F protein ecto-domain polypeptide and one or moreother RSV proteins (e.g., a G protein and/or an M protein) and/or it maybe combined with immunogens from other pathogens.

The composition may include preservatives such as thiomersal or2-phenoxyethanol. It is preferred, however, that the vaccine should besubstantially free from (i.e., less than 5 μg/ml) mercurial material,e.g., thiomersal-free. Immunogenic compositions containing no mercuryare more preferred. Preservative-free immunogenic compositions areparticularly preferred.

To control tonicity, it is preferred to include a physiological salt,such as a sodium salt. Sodium chloride (NaCl) is preferred, which may bepresent at between 1 and 20 mg/ml. Other salts that may be presentinclude potassium chloride, potassium dihydrogen phosphate, disodiumphosphate dehydrate, magnesium chloride, calcium chloride, and the like.

Compositions will generally have an osmolality of between 200 mOsm/kgand 400 mOsm/kg, preferably between 240-360 mOsm/kg, and will morepreferably fall within the range of 290-310 mOsm/kg.

Compositions may include one or more buffers. Typical buffers include: aphosphate buffer; a Tris buffer; a borate buffer; a succinate buffer; ahistidine buffer (particularly with an aluminum hydroxide adjuvant); ora citrate buffer. Buffers will typically be included in the 5-20 mMrange. The pH of a composition will generally be between 5.0 and 8.1,and more typically between 6.0 and 8.0, e.g., between 6.5 and 7.5, orbetween 7.0 and 7.8. A process of the invention may therefore include astep of adjusting the pH of the bulk vaccine prior to packaging.

The composition is preferably sterile. The composition is preferablynon-pyrogenic, e.g., containing <1 EU (endotoxin unit, a standardmeasure) per dose, and preferably <0.1 EU per dose. The composition ispreferably gluten free. Human vaccines are typically administered in adosage volume of about 0.5 ml, although a half dose (i.e., about 0.25ml) may be administered to children.

Adjuvants

Compositions of the invention, that contain RSV-F polypeptids, ornucleic acids that encode RSV-F polypeptids, may also include one ormore adjuvants, for example two, three, four or more adjuvants, whichcan function to enhance the immune responses (humoral and/or cellular)elicited in a patient who receives the composition. The adjuvants mayinclude a TH1 adjuvant and/or a TH2 adjuvant. Adjuvants which may beused in compositions of the invention include, but are not limited to:

-   -   Mineral-containing compositions. Mineral-containing compositions        suitable for use as adjuvants in the invention include mineral        salts, such as calcium salts and aluminum salts (or mixtures        thereof). The invention includes mineral salts such as        hydroxides (e.g. oxyhydroxides), phosphates (e.g.        hydroxyphosphates, orthophosphates), sulphates, etc., or        mixtures of different mineral compounds, with the compounds        taking any suitable form (e.g. gel, crystalline, amorphous,        etc.), and with adsorption being preferred. Calcium salts        include calcium phosphate (e.g., the “CAP” particles disclosed        in ref. 38). Aluminum salts include hydroxides, phosphates,        sulfates, and the like. The mineral containing compositions may        also be formulated as a particle of metal salt (39). Aluminum        salt adjuvants are described in more detail below.    -   Oil emulsion compositions (see in more detail below). Oil        emulsion compositions suitable for use as adjuvants in the        invention include squalene-water emulsions, such as MF59 (5%        Squalene, 0.5% Tween 80 and 0.5% Span, formulated into submicron        particles using a microfluidizer).    -   Cytokine-inducing agents (see in more detail below).        Cytokine-inducing agents suitable for use in the invention        include toll-like receptor 7 (TLR7) agonists (e.g.        benzonaphthyridine compounds disclosed in WO 2009/111337.    -   Saponins (chapter 22 of ref. 74), which are a heterologous group        of sterol glycosides and triterpenoid glycosides that are found        in the bark, leaves, stems, roots and even flowers of a wide        range of plant species. Saponin from the bark of the Quillaia        saponaria Molina tree have been widely studied as adjuvants.        Saponin can also be commercially obtained from Smilax ornata        (sarsaprilla), Gypsophilla paniculata (brides veil), and        Saponaria officianalis (soap root). Saponin adjuvant        formulations include purified formulations, such as QS21, as        well as lipid formulations, such as ISCOMs. QS21 is marketed as        STIMULON™. Saponin compositions have been purified using HPLC        and RP-HPLC. Specific purified fractions using these techniques        have been identified, including QS7, QS17, QS18, QS21, QH-A,        QH-B and QH-C. Preferably, the saponin is QS21. A method of        production of QS21 is disclosed in ref. 40. Saponin formulations        may also comprise a sterol, such as cholesterol (41).        Combinations of saponins and cholesterols can be used to form        unique particles called immunostimulating complexes (ISCOMs)        (chapter 23 of ref. 74). ISCOMs typically also include a        phospholipid such as phosphatidylethanolamine or        phosphatidylcholine. Any known saponin can be used in ISCOMs.        Preferably, the ISCOM includes one or more of QuilA, QHA & QHC.        ISCOMs are further described in refs. 41-43. Optionally, the        ISCOMS may be devoid of additional detergent (44). A review of        the development of saponin based adjuvants can be found in refs.        45 & 46.    -   Fatty adjuvants (see in more detail below), including        oil-in-water emulsions, modified natural lipid As derived from        enterobacterial lipopolysaccharides, phospholipid compounds        (such as the synthetic phospholipid dimer, E6020) and the like.    -   Bacterial ADP-ribosylating toxins (e.g., the E. coli heat labile        enterotoxin “LT”, cholera toxin “CT”, or pertussis toxin “PT”)        and detoxified derivatives thereof, such as the mutant toxins        known as LT-K63 and LT-R72 (47). The use of detoxified        ADP-ribosylating toxins as mucosal adjuvants is described in        ref. 48 and as parenteral adjuvants in ref. 49.    -   Bioadhesives and mucoadhesives, such as esterified hyaluronic        acid microspheres (50) or chitosan and its derivatives (51).    -   Microparticles (i.e., a particle of ˜100 nm to ˜150 μm in        diameter, more preferably ˜200 nm to ˜30 μm in diameter, or ˜500        nm to ˜10 μm in diameter) formed from materials that are        biodegradable and non-toxic (e.g., a poly(α-hydroxy acid), a        polyhydroxybutyric acid, a polyorthoester, a polyanhydride, a        polycaprolactone, and the like), with poly(lactide-co-glycolide)        being preferred,        optionally treated to have a negatively-charged surface (e.g.,        with SDS) or a positively-charged surface (e.g., with a cationic        detergent, such as CTAB).    -   Liposomes (Chapters 13 & 14 of ref. 74). Examples of liposome        formulations suitable for use as adjuvants are described in        refs. 52-54.    -   Polyoxyethylene ethers and polyoxyethylene esters (55). Such        formulations further include polyoxyethylene sorbitan ester        surfactants in combination with an octoxynol (56) as well as        polyoxyethylene alkyl ethers or ester surfactants in combination        with at least one additional non-ionic surfactant such as an        octoxynol (57). Preferred polyoxyethylene ethers are selected        from the following group: polyoxyethylene-9-lauryl ether        (laureth 9), polyoxyethylene-9-steoryl ether,        polyoxytheylene-8-steoryl ether, polyoxyethylene-4-lauryl ether,        polyoxyethylene-35-lauryl ether, and polyoxyethylene-23-lauryl        ether.    -   Muramyl peptides, such as        N-acetylmuramyl-L-threonyl-D-isoglutamine (“thr-MDP”),        N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP),        N-acetylglucsaminyl-N-acetylmuramyl-L-Al-D-isoglu-L-Ala-dipalmitoxy        propylamide (“DTP-DPP”, or “Theramide™),        N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine        (“MTP-PE”).    -   An outer membrane protein proteosome preparation prepared from a        first Gram-negative bacterium in combination with a        liposaccharide preparation derived from a second Gram-negative        bacterium, wherein the outer membrane protein proteosome and        liposaccharide preparations form a stable non-covalent adjuvant        complex. Such complexes include “IVX-908”, a complex comprised        of Neisseria meningitidis outer membrane and        lipopolysaccharides.    -   A polyoxidonium polymer (58, 59) or other N-oxidized        polyethylene-piperazine derivative.    -   Methyl inosine 5′-monophosphate (“MIMP”) (60).    -   A polyhydroxlated pyrrolizidine compound (61), such as one        having formula:

-   -    where R is selected from the group comprising hydrogen,        straight or branched, unsubstituted or substituted, saturated or        unsaturated acyl, alkyl (e.g., cycloalkyl), alkenyl, alkynyl and        aryl groups, or a pharmaceutically acceptable salt or derivative        thereof. Examples include, but are not limited to: casuarine,        casuarine-6-α-D-glucopyranose, 3-epi-casuarine, 7-epi-casuarine,        3,7-diepi-casuarine, and the like    -   A CD1d ligand, such as an α-glycosylceramide (62-69) (e.g.,        α-galactosylceramide), phytosphingosine-containing        α-glycosylceramides, OCH, KRN7000        [(2S,3S,4R)-1-O-(α-D-galactopyranosyl)-2-(N-hexacosanoylamino)-1,3,4-octadecanetriol],        CRONY-101, 3″-O-sulfo-galactosylceramide, etc.    -   A gamma inulin (70) or derivative thereof, such as algammulin.    -   Virosomes and virus-like particles (VLPs). These structures        generally contain one or more proteins from a virus optionally        combined or formulated with a phospholipid. They are generally        non-pathogenic, non-replicating and generally do not contain any        of the native viral genome. The viral proteins may be        recombinantly produced or isolated from whole viruses. These        viral proteins suitable for use in virosomes or VLPs include        proteins derived from influenza virus (such as HA or NA),        Hepatitis B virus (such as core or capsid proteins), Hepatitis E        virus, measles virus, Sindbis virus, Rotavirus, Foot-and-Mouth        Disease virus, Retrovirus, Norwalk virus, human Papilloma virus,        HIV, RNA-phages, Qβ-phage (such as coat proteins), GA-phage,        fr-phage, AP205 phage, and Ty (such as retrotransposon Ty        protein p1).

These and other adjuvant-active substances are discussed in more detailin references 74 & 75.

Compositions may include two, three, four or more adjuvants. Forexample, compositions of the invention may advantageously include bothan oil-in-water emulsion and a cytokine-inducing agent, or both amineral-containing composition and a cytokine-inducing agent, or twooil-in-water emulsion adjuvants, or two benzonaphthyridine compounds,etc.

Antigens and adjuvants in a composition will typically be in admixture.

Oil Emulsion Adjuvants

Oil emulsion compositions suitable for use as adjuvants in the inventioninclude squalene-water emulsions, such as MF59 (5% Squalene, 0.5% Tween80, and 0.5% Span 85, formulated into submicron particles using amicrofluidizer). Complete Freund's adjuvant (CFA) and incompleteFreund's adjuvant (IFA) may also be used.

Various oil-in-water emulsions are known, and they typically include atleast one oil and at least one surfactant, with the oil(s) andsurfactant(s) being biodegradable (metabolizable) and biocompatible. Theoil droplets in the emulsion are generally less than 5 μm in diameter,and may even have a sub-micron diameter, with these small sizes beingachieved with a microfluidizer to provide stable emulsions. Dropletswith a size less than 220 nm are preferred as they can be subjected tofilter sterilization.

The invention can be used with oils such as those from an animal (suchas fish) or vegetable source. Sources for vegetable oils include nuts,seeds and grains. Peanut oil, soybean oil, coconut oil, and olive oil,the most commonly available, exemplify the nut oils. Jojoba oil can beused, e.g., obtained from the jojoba bean. Seed oils include saffloweroil, cottonseed oil, sunflower seed oil, sesame seed oil and the like.In the grain group, corn oil is the most readily available, but the oilof other cereal grains such as wheat, oats, rye, rice, teff, triticaleand the like may also be used. 6-10 carbon fatty acid esters of glyceroland 1,2-propanediol, while not occurring naturally in seed oils, may beprepared by hydrolysis, separation and esterification of the appropriatematerials starting from the nut and seed oils. Fats and oils frommammalian milk are metabolizable and may therefore be used in thepractice of this invention. The procedures for separation, purification,saponification and other means necessary for obtaining pure oils fromanimal sources are well known in the art. Most fish containmetabolizable oils which may be readily recovered. For example, codliver oil, shark liver oils, and whale oil such as spermaceti exemplifyseveral of the fish oils which may be used herein. A number of branchedchain oils are synthesized biochemically in 5-carbon isoprene units andare generally referred to as terpenoids. Shark liver oil contains abranched, unsaturated terpenoid known as squalene,2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexaene, which isparticularly preferred herein. Squalane, the saturated analog tosqualene, is also preferred oil. Fish oils, including squalene andsqualane, are readily available from commercial sources or may beobtained by methods known in the art. Other preferred oils are thetocopherols (see below). Mixtures of oils can be used.

Surfactants can be classified by their ‘HLB’ (hydrophile/lipophilebalance). Preferred surfactants of the invention have a HLB of at least10, preferably at least 15, and more preferably at least 16. Theinvention can be used with surfactants including, but not limited to:the polyoxyethylene sorbitan esters surfactants (commonly referred to asthe Tweens), especially polysorbate 20 and polysorbate 80; copolymers ofethylene oxide (EO), propylene oxide (PO), and/or butylene oxide (BO),sold under the DOWFAX™ tradename, such as linear EO/PO block copolymers;octoxynols, which can vary in the number of repeating ethoxy(oxy-1,2-ethanediyl) groups, with octoxynol-9 (Triton X-100, ort-octylphenoxypolyethoxyethanol) being of particular interest;(octylphenoxy)polyethoxyethanol (IGEPAL CA-630/NP-40); phospholipidssuch as phosphatidylcholine (lecithin); nonylphenol ethoxylates, such asthe TERGITOL™ NP series; polyoxyethylene fatty ethers derived fromlauryl, cetyl, stearyl and oleyl alcohols (known as Brij surfactants),such as triethyleneglycol monolauryl ether (Brij 30); and sorbitanesters (commonly known as the SPANs), such as sorbitan trioleate (Span85) and sorbitan monolaurate. Non-ionic surfactants are preferred.Preferred surfactants for including in the emulsion are TWEEN 80™(polyoxyethylene sorbitan monooleate), Span 85 (sorbitan trioleate),lecithin and Triton X-100.

Mixtures of surfactants can be used e.g., TWEEN 80™/Span 85 mixtures. Acombination of a polyoxyethylene sorbitan ester such as polyoxyethylenesorbitan monooleate (TWEEN 80™) and an octoxynol such ast-octylphenoxypolyethoxyethanol (Triton X-100) is also suitable. Anotheruseful combination comprises laureth 9 plus a polyoxyethylene sorbitanester and/or an octoxynol.

Preferred amounts of surfactants (% by weight) are: polyoxyethylenesorbitan esters (such as TWEEN 80™) 0.01 to 1%, in particular about0.1%; octyl- or nonylphenoxy polyoxyethanols (such as Triton X-100, orother detergents in the Triton series) 0.001 to 0.1%, in particular0.005 to 0.02%; polyoxyethylene ethers (such as laureth 9) 0.1 to 20%,preferably 0.1 to 10% and in particular 0.1 to 1% or about 0.5%.

Specific oil-in-water emulsion adjuvants useful with the inventioninclude, but are not limited to:

-   -   A submicron emulsion of squalene, TWEEN 80™, and Span 85. The        composition of the emulsion by volume can be about 5% squalene,        about 0.5% polysorbate 80 and about 0.5% Span 85. In weight        terms, these ratios become 4.3% squalene, 0.5% polysorbate 80        and 0.48% Span 85. This adjuvant is known as ‘MF59’ (71-73), as        described in more detail in Chapter 10 of ref. 74 and chapter 12        of ref. 75. The MF59 emulsion advantageously includes citrate        ions, e.g., 10 mM sodium citrate buffer.    -   An emulsion of squalene, a tocopherol, and TWEEN 80™. The        emulsion may include phosphate buffered saline. It may also        include Span 85 (e.g., at 1%) and/or lecithin. These emulsions        may have from 2 to 10% squalene, from 2 to 10% tocopherol and        from 0.3 to 3% TWEEN 80™, and the weight ratio of        squalene:tocopherol is preferably <1 as this provides a more        stable emulsion. Squalene and TWEEN 80™ may be present volume        ratio of about 5:2. One such emulsion can be made by dissolving        TWEEN 80™ in PBS to give a 2% solution, then mixing 90 ml of        this solution with a mixture of (5 g of DL-α-tocopherol and 5 ml        squalene), then microfluidizing the mixture. The resulting        emulsion may have submicron oil droplets, e.g., with an average        diameter of between 100 and 250 nm, preferably about 180 nm.    -   An emulsion of squalene, a tocopherol, and a Triton detergent        (e.g., Triton X-100). The emulsion may also include a 3d-MPL        (see below). The emulsion may contain a phosphate buffer.    -   An emulsion comprising a polysorbate (e.g., polysorbate 80), a        Triton detergent (e.g., Triton X-100) and a tocopherol (e.g., an        α-tocopherol succinate). The emulsion may include these three        components at a mass ratio of about 75:11:10 (e.g., 750 μg/ml        polysorbate 80, 110 μg/ml Triton X-100 and 100 μg/ml        α-tocopherol succinate), and these concentrations should include        any contribution of these components from antigens. The emulsion        may also include squalene. The emulsion may also include a        3d-MPL (see below). The aqueous phase may contain a phosphate        buffer.    -   An emulsion of squalane, polysorbate 80 and poloxamer 401        (“PLURONIC™ L121”). The emulsion can be formulated in phosphate        buffered saline, pH 7.4. This emulsion is a useful delivery        vehicle for muramyl dipeptides, and has been used with        threonyl-MDP in the “SAF-1” adjuvant (76) (0.05-1% Thr-MDP, 5%        squalane, 2.5% Pluronic L121 and 0.2% polysorbate 80). It can        also be used without the Thr-MDP, as in the “AF” adjuvant (77)        (5% squalane, 1.25% Pluronic L121 and 0.2% polysorbate 80).        Microfluidization is preferred.    -   An emulsion comprising squalene, an aqueous solvent, a        polyoxyethylene alkyl ether hydrophilic nonionic surfactant        (e.g. polyoxyethylene (12) cetostearyl ether) and a hydrophobic        nonionic surfactant (e.g. a sorbitan ester or mannide ester,        such as sorbitan monoleate or ‘Span 80’). The emulsion is        preferably thermoreversible and/or has at least 90% of the oil        droplets (by volume) with a size less than 200 nm. The emulsion        may also include one or more of: alditol; a cryoprotective agent        (e.g. a sugar, such as dodecylmaltoside and/or sucrose); and/or        an alkylpolyglycoside. Such emulsions may be lyophilized.    -   An emulsion having from 0.5-50% of an oil, 0.1-10% of a        phospholipid, and 0.05-5% of a non-ionic surfactant. As        described in reference 78, preferred phospholipid components are        phosphatidylcholine, phosphatidylethanolamine,        phosphatidylserine, phosphatidylinositol, phosphatidylglycerol,        phosphatidic acid, sphingomyelin and cardiolipin. Submicron        droplet sizes are advantageous.    -   A submicron oil-in-water emulsion of a non-metabolizable oil        (such as light mineral oil) and at least one surfactant (such as        lecithin, TWEEN 80™ or Span 80). Additives may be included, such        as QuilA saponin, cholesterol, a saponin-lipophile conjugate        (such as GPI-0100, described in reference 79, produced by        addition of aliphatic amine to desacylsaponin via the carboxyl        group of glucuronic acid), dimethyidioctadecylammonium bromide        and/or N,N-dioctadecyl-N,N-bis (2-hydroxyethyl)propanediamine.    -   An emulsion comprising a mineral oil, a non-ionic lipophilic        ethoxylated fatty alcohol, and a non-ionic hydrophilic        surfactant (e.g. an ethoxylated fatty alcohol and/or        polyoxyethylene-polyoxypropylene block copolymer).    -   An emulsion comprising a mineral oil, a non-ionic hydrophilic        ethoxylated fatty alcohol, and a non-ionic lipophilic surfactant        (e.g. an ethoxylated fatty alcohol and/or        polyoxyethylene-polyoxypropylene block copolymer).    -   An emulsion in which a saponin (e.g., QuilA or QS21) and a        sterol (e.g., a cholesterol) are associated as helical micelles        (80).

The emulsions may be mixed with antigen extemporaneously, at the time ofdelivery. Thus the adjuvant and antigen may be kept separately in apackaged or distributed vaccine, ready for final formulation at the timeof use. The antigen will generally be in an aqueous form, such that thevaccine is finally prepared by mixing two liquids. The volume ratio ofthe two liquids for mixing can vary (e.g., between 5:1 and 1:5) but isgenerally about 1:1.

Cytokine-Inducing Agents

Cytokine-inducing agents for inclusion in compositions of the inventionare able, when administered to a patient, to elicit the immune system torelease cytokines, including interferons and interleukins. Preferredagents can elicit the release of one or more of: interferon-γ;interleukin-1; interleukin-2; interleukin-12; TNF-α; TNF-β; and GM-CSF.Preferred agents elicit the release of cytokines associated with aTh1-type immune response, e.g., interferon-γ, TNF-α, interleukin-2.Stimulation of both interferon-γ and interleukin-2 is preferred.

As a result of receiving a composition of the invention, therefore, apatient will have T cells that, when stimulated with a RSV F protein,will release the desired cytokine(s) in an antigen-specific manner. Forexample, T cells purified from their blood will release γ-interferonwhen exposed in vitro to F protein. Methods for measuring such responsesin peripheral blood mononuclear cells (PBMC) are known in the art, andinclude ELISA, ELISPOT, flow-cytometry and real-time PCR. For example,reference 81 reports a study in which antigen-specific T cell-mediatedimmune responses against tetanus toxoid, specifically γ-interferonresponses, were

monitored, and found that ELISPOT was the most sensitive method todiscriminate antigen-specific TT-induced responses from spontaneousresponses, but that intracytoplasmic cytokine detection by flowcytometry was the most efficient method to detect re-stimulatingeffects.

Suitable cytokine-inducing agents include, but are not limited to:

-   -   An immunostimulatory oligonucleotide, such as one containing a        CpG motif (a dinucleotide sequence containing an unmethylated        cytosine linked by a phosphate bond to a guanosine), or a        double-stranded RNA, or an oligonucleotide containing a        palindromic sequence, or an oligonucleotide containing a        poly(dG) sequence.    -   3-O-deacylated monophosphoryl lipid A (‘3dMPL’, also known as        ‘MPL™’) (82-85).    -   An imidazoquinoline compound, such as IMIQUIMOD™ (“R-837”) (86,        87), RESIQUIMOD™ (“R-848”) (88), and their analogs; and salts        thereof (e.g., the hydrochloride salts). Further details about        immunostimulatory imidazoquinolines can be found in references        89 to 93.    -   A benzonaphthyridine compound, such as: (a) a compound having        the formula:

-   -   wherein:        -   R⁴ is selected from H, halogen, —C(O)OR⁷, —C(O)R⁷,            —C(O)N(R¹¹R¹²), —N(R¹¹R¹²), —N(R⁹)₂, —NHN(R⁹)₂, —SR⁷,            —(CH₂)_(n)OR⁷, —(CH₂)_(n)R⁷, -LR⁸, -LR¹⁰, —OLR⁸, —OLR¹⁰,            C₁-C₆alkyl, C₁-C₆heteroalkyl, C₁-C₆haloalkyl, C₂-C₈alkene,            C₂-C₈alkyne, C₁-C₆alkoxy, C₁-C₆haloalkoxy, aryl, heteroaryl,            C₃-C₈cycloalkyl, and C₃-C₈heterocycloalkyl, wherein the            C₁-C₆alkyl, C₁-C₆heteroalkyl, C₁-C₆haloalkyl, C₂-C₈alkene,            C₂-C₈alkyne, C₁-C₆alkoxy, C₁-C₆haloalkoxy, aryl, heteroaryl,            C₃-C₈cycloalkyl, and C₃-C₈heterocycloalkyl groups of R⁴ are            each optionally substituted with 1 to 3 substituents            independently selected from halogen, —CN, —NO₂, —R⁷, —OR⁸,            —C(O)R⁸, —OC(O)R⁸, —C(O)OR⁸, —N(R⁹)₂, —P(O)(OR⁸)₂,            —OP(O)(OR⁸)₂, —P(O)(OR¹⁰)₂,            -   —OP(O)(OR¹⁰)₂, —C(O)N(R⁹)₂, —S(O)₂R⁸, —S(O)R⁸,                —S(O)₂N(R⁹)₂, and —NR⁹S(O)₂R⁸;        -   each L is independently selected from a bond,            —(O(CH₂)_(m))_(t)—, C₁-C₆alkyl, C₂-C₆alkenylene and            C₂-C₆alkynylene, wherein the C₁-C₆alkyl, C₂-C₆alkenylene and            C₂-C₆alkynylene of L are each optionally substituted with 1            to 4 substituents independently selected from halogen, —R⁸,            —OR⁸, -N(R⁹)₂, —P(O)(OR⁸)₂, —OP(O)(OR⁸)₂,            -   —P(O)(OR¹⁰)₂, and —OP(O)(OR¹⁰)₂;        -   R⁷ is selected from H, C₁-C₆alkyl, aryl, heteroaryl,            C₃-C₈cycloalkyl, C₁-C₆heteroalkyl, C₁-C₆haloalkyl,            C₂-C₈alkene, C₂-C₈alkyne, C₁-C₆alkoxy, C₁-C₆haloalkoxy, and            C₃-C₈heterocycloalkyl, wherein the C₁-C₆alkyl, aryl,            heteroaryl, C₃-C₈cycloalkyl,            -   C₁-C₆heteroalkyl, C₁-C₆haloalkyl, C₂-C₈alkene,                C₂-C₈alkyne, C₁-C₆alkoxy, C₁-C₆haloalkoxy, and                C₃-C₈heterocycloalkyl groups of R⁷ are each optionally                substituted with 1 to 3 R¹³ groups;        -   each R⁸ is independently selected from H, —CH(R¹⁰)₂,            C₁-C₈alkyl, C₂-C₈alkene, C₂-C₈alkyne, C₁-C₆haloalkyl,            C₁-C₆alkoxy, C₁-C₆heteroalkyl, C₃-C₈cycloalkyl,            C₂-C₈heterocycloalkyl, C₁-C₆hydroxyalkyl and            C₁-C₆haloalkoxy, wherein the C₁-C₈alkyl, C₂-C₈alkene,            C₂-C₈alkyne, C₁-C₆heteroalkyl, C₁-C₆haloalkyl, C₁-C₆alkoxy,            C₃-C₈cycloalkyl, C₂-C₈heterocycloalkyl, C₁-C₆hydroxyalkyl            and C₁-C₆haloalkoxy groups of R⁸ are each optionally            substituted with 1 to 3 substituents independently selected            from —CN, R¹¹, —OR¹¹, —SR¹¹, —C(O)R¹¹, —OC(O)R¹¹,            -   —C(O)N(R⁹)₂, —C(O)OR¹¹, —NR⁹C(O)R¹¹, —NR⁹R¹⁰, —NR¹¹R¹²,                —N(R⁹)₂, —OR⁹, —OR¹⁰, —C(O)NR¹¹R¹², —C(O)NR¹¹OH,                —S(O)₂R¹¹, —S(O)R¹¹, —S(O)₂NR¹¹R¹², —NR¹¹S(O)₂R¹¹,                —P(O)(OR¹¹)₂, and —OP(O)(OR¹¹)₂;        -   each R⁹ is independently selected from H, —C(O)R⁸, —C(O)OR⁸,            —C(O)R¹⁰, —C(O)OR¹⁰, —S(O)₂R¹⁰, —C₁-C₆ alkyl, C₁-C₆            heteroalkyl and C₃-C₆ cycloalkyl, or each R⁹ is            independently a C₁-C₆alkyl that together with N they are            attached to form a C₃-C₈heterocycloalkyl, wherein the            C₃-C₈heterocycloalkyl ring optionally contains an additional            heteroatom selected from N, O and S, and wherein the C₁-C₆            alkyl, C₁-C₆ heteroalkyl, C₃-C₆ cycloalkyl, or            -   C₃-C₈heterocycloalkyl groups of R⁹ are each optionally                substituted with 1 to 3 substituents independently                selected from            -   —CN, R¹¹, —OR¹¹, —SR¹¹, —C(O)R¹¹, —OC(O)R¹¹, —C(O)OR¹¹,            -   —NR¹¹R¹², —C(O)NR¹¹R¹², —C(O)NR¹¹OH, —S(O)₂R¹¹,                —S(O)R¹¹, —S(O)₂NR¹¹R¹², —NR¹¹S(O)₂R¹¹, —P(O)(OR¹¹)₂,                and            -   —OP(O)(OR¹¹)₂;        -   each R¹⁰ is independently selected from aryl,            C₃-C₈cycloalkyl, C₃-C₈heterocycloalkyl and heteroaryl,            wherein the aryl, C₃-C₈cycloalkyl, C₃-C₈heterocycloalkyl and            heteroaryl groups are optionally substituted with 1 to 3            substituents selected from halogen, —R⁸, —OR⁸, -LR⁹, -LOR⁹,            —N(R⁹)₂, —NR⁹C(O)R⁸, —NR⁹CO₂R⁸, —CO₂R⁸, —C(O)R⁸ and            —C(O)N(R⁹)₂;        -   R¹¹ and R¹² are independently selected from H, C₁-C₆alkyl,            C₁-C₆heteroalkyl, C₁-C₆haloalkyl, aryl, heteroaryl,            C₃-C₈cycloalkyl, and C₃-C₈heterocycloalkyl, wherein the            C₁-C₆alkyl, C₁-C₆heteroalkyl, C₁-C₆haloalkyl, aryl,            heteroaryl, C₃-C₈cycloalkyl, and C₃-C₈heterocycloalkyl            groups of R¹¹ and R¹² are each optionally substituted with 1            to 3 substituents independently selected from halogen, —CN,            R⁸, —OR⁸, —C(O)R⁸,            -   ⁻OC(O)R⁸, —C(O)OR⁸, —N(R⁹)₂, —NR⁸C(O)R⁸, —NR⁸C(O)OR⁸,                —C(O)N(R⁹)₂, C₃-C₈heterocycloalkyl, —S(O)₂R⁸,                —S(O)₂N(R⁹)₂, —NR⁹S(O)₂R⁸, C₁-C₆haloalkyl and                C₁-C₆haloalkoxy;        -   or R¹¹ and R¹² are each independently C₁-C₆alkyl and taken            together with the N atom to which they are attached form an            optionally substituted C₃-C₈heterocycloalkyl ring optionally            containing an additional heteroatom selected from N, O and            S;        -   each R¹³ is independently selected from halogen, —CN, -LR⁹,            -LOR⁹, —OLR⁹, -LR¹⁰, -LOR¹⁰, —OLR¹⁰, -LR⁸, -LOR⁸, —OLR⁸,            -LSR⁸, -LSR¹⁰, -LC(O)R⁸, —OLC(O)R⁸, -LC(O)OR⁸, -LC(O)R¹⁰,            -LOC(O)OR⁸, -LC(O)NR⁹R¹¹, -LC(O)NR⁹R⁸, -LN(R⁹)₂, -LNR⁹R⁸,            -LNR⁹R¹⁰, -LC(O)N(R⁹)₂, -LS(O)₂R⁸, -LS(O)R⁸, -LC(O)NR⁸OH,            -LNR⁹C(O)R⁸, -LNR⁹C(O)OR⁸, -LS(O)₂N(R⁹)₂, —OLS(O)₂N(R⁹)₂,            -LNR⁹S(O)₂R⁸, -LC(O)NR⁹LN(R⁹)₂, -LP(O)(OR⁸)₂, -LOP(O)(OR⁸)₂,            -LP(O)(OR¹⁰)₂ and -OLP(O)(OR¹⁰)₂;        -   each R^(A) is independently selected from —R⁸, —R⁷, —OR⁷,            —OR⁸, —R¹⁰, —OR¹⁰, —SR⁸, —NO₂, —CN, —N(R⁹)₂, —NR⁹C(O)R⁸,            —NR⁹C(S)R⁸, —NR⁹C(O)N(R⁹)₂, —NR⁹C(S)N(R⁹)₂, —NR⁹CO₂R⁸,            —NR⁹NR⁹C(O)R⁸, —NR⁹NR⁹C(O)N(R⁹)₂, —NR⁹NR⁹CO₂R⁸, —C(O)C(O)R⁸,            —C(O)CH₂C(O)R⁸, —CO₂R⁸, —(CH₂)_(n)CO₂R⁸, —C(O)R⁸, —C(S)R⁸,            —C(O)N(R⁹)₂, —C(S)N(R⁹)₂, —OC(O)N(R⁹)₂, —OC(O)R⁸,            —C(O)N(OR⁸)R⁸, —C(NOR⁸)R⁸, —S(O)₂R⁸, —S(O)₃R⁸, —SO₂N(R⁹)₂,            —S(O)R⁸, —NR⁹SO₂N(R⁹)₂, —NR⁹SO₂R⁸, —P(O)(OR⁸)₂,            —OP(O)(OR⁸)₂, —P(O)(OR¹⁰)₂, —OP(O)(OR¹⁰)₂, —N(OR⁸)R⁸,            —CH═CHCO₂R⁸, —C(═NH)—N(R⁹)₂, and —(CH₂)_(n)NHC(O)R⁸; or two            adjacent R^(A) substituents on Ring A form a 5-6 membered            ring that contains up to two heteroatoms as ring members;        -   n is, independently at each occurrence, 0, 1, 2, 3, 4, 5, 6,            7 or 8;        -   each m is independently selected from 1, 2, 3, 4, 5 and 6,            and        -   t is 1, 2, 3, 4, 5, 6, 7 or 8; (b) a compound having the            formula:

-   -   wherein:        -   R⁴ is selected from H, halogen, —C(O)OR⁷, —C(O)R⁷,            —C(O)N(R¹¹R¹²), —N(R¹¹R¹²), —N(R⁹)₂, —NHN(R⁹)₂, —SR⁷,            —(CH₂)_(n)OR⁷, —(CH₂)_(n)R⁷, -LR⁸, -LR¹⁰, —OLR⁸, —OLR¹⁰,            C₁-C₆alkyl, C₁-C₆heteroalkyl, C₁-C₆haloalkyl, C₂-C₈alkene,            C₂-C₈alkyne, C₁-C₆alkoxy, C₁-C₆haloalkoxy, aryl, heteroaryl,            C₃-C₈cycloalkyl, and C₃-C₈heterocycloalkyl, wherein the            C₁-C₆alkyl, C₁-C₆heteroalkyl, C₁-C₆haloalkyl, C₂-C₈alkene,            C₂-C₈alkyne, C₁-C₆alkoxy, C₁-C₆haloalkoxy, aryl, heteroaryl,            C₃-C₈cycloalkyl, and C₃-C₈heterocycloalkyl groups of R⁴ are            each optionally substituted with 1 to 3 substituents            independently selected from halogen, —CN, —NO₂, —R⁷, —OR⁸,            —C(O)R⁸, —OC(O)R⁸, —C(O)OR⁸, —N(R⁹)₂, —P(O)(OR⁸)₂,            —OP(O)(OR⁸)₂, —P(O)(OR¹⁰)₂,            -   —OP(O)(OR¹⁰)₂, —C(O)N(R⁹)₂, —S(O)₂R⁸, —S(O)R⁸,            -   —S(O)₂N(R⁹)₂, and —NR⁹S(O)₂R⁸;        -   each L is independently selected from a bond,            —(O(CH₂)_(m))_(t)—, C₁-C₆alkyl, C₂-C₆alkenylene and            C₂-C₆alkynylene, wherein the C₁-C₆alkyl, C₂-C₆alkenylene and            C₂-C₆alkynylene of L are each optionally substituted with 1            to 4 substituents independently selected from halogen, —R⁸,            —OR⁸, —N(R⁹)₂, —P(O)(OR⁸)₂, —OP(O)(OR⁸)₂,            -   —P(O)(OR¹⁰)₂, and —OP(O)(OR¹⁰)₂;        -   R⁷ is selected from H, C₁-C₆alkyl, aryl, heteroaryl,            C₃-C₈cycloalkyl, C₁-C₆heteroalkyl, C₁-C₆haloalkyl,            C₂-C₈alkene, C₂-C₈alkyne, C₁-C₆alkoxy, C₁-C₆haloalkoxy, and            C₃-C₈heterocycloalkyl, wherein the C₁-C₆alkyl, aryl,            heteroaryl, C₃-C₈cycloalkyl,            -   C₁-C₆heteroalkyl, C₁-C₆haloalkyl, C₂-C₈alkene,                C₂-C₈alkyne, C₁-C₆alkoxy, C₁-C₆haloalkoxy, and                C₃-C₈heterocycloalkyl groups of R⁷ are each optionally                substituted with 1 to 3 R¹³ groups;        -   each R⁸ is independently selected from H, —CH(R¹⁰)₂,            C₁-C₈alkyl, C₂-C₈alkene, C₂-C₈alkyne, C₁-C₆haloalkyl,            C₁-C₆alkoxy, C₁-C₆heteroalkyl, C₃-C₈cycloalkyl,            C₂-C₈heterocycloalkyl, C₁-C₆hydroxyalkyl and            C₁-C₆haloalkoxy, wherein the C₁-C₈alkyl, C₂-C₈alkene,            C₂-C₈alkyne, C₁-C₆heteroalkyl, C₁-C₆haloalkyl, C₁-C₆alkoxy,            C₃-C₈cycloalkyl, C₂-C₈heterocycloalkyl, C₁-C₆hydroxyalkyl            and C₁-C₆haloalkoxy groups of R⁸ are each optionally            substituted with 1 to 3 substituents independently selected            from —CN, R¹¹, —OR¹¹, —SR¹¹, —C(O)R¹¹, —OC(O)R¹¹,            -   —C(O)N(R⁹)₂, —C(O)OR¹¹, —NR⁹C(O)R¹¹, —NR⁹R¹⁰, —NR¹¹R¹²,                —N(R⁹)₂, —OR⁹, —OR¹⁰, —C(O)NR¹¹R¹², —C(O)NR¹¹OH,                —S(O)₂R¹¹, —S(O)R¹¹, —S(O)₂NR¹¹R¹², —NR¹¹S(O)₂R¹¹,                —P(O)(OR¹¹)₂, and —OP(O)(OR¹¹)₂;        -   each R⁹ is independently selected from H, —C(O)R⁸, —C(O)OR⁸,            —C(O)R¹⁰, —C(O)OR¹⁰, —S(O)₂R¹⁰, —C₁-C₆ alkyl, C₁-C₆            heteroalkyl and C₃-C₆ cycloalkyl, or each R⁹ is            independently a C₁-C₆alkyl that together with N they are            attached to form a C₃-C₈heterocycloalkyl, wherein the            C₃-C₈heterocycloalkyl ring optionally contains an additional            heteroatom selected from N, O and S, and wherein the C₁-C₆            alkyl, C₁-C₆ heteroalkyl, C₃-C₆ cycloalkyl, or            -   C₃-C₈heterocycloalkyl groups of R⁹ are each optionally                substituted with 1 to 3 substituents independently                selected from            -   —CN, R¹¹, —OR¹¹, —SR¹¹, —C(O)R¹¹, —OC(O)R¹¹, —C(O)OR¹¹,                —NR¹¹R¹², —C(O)NR¹¹R¹², —C(O)NR¹¹OH, —S(O)₂R¹¹,                —S(O)R¹¹, —S(O)₂NR¹¹R¹², —NR¹¹S(O)₂R¹¹, —P(O)(OR¹¹)₂,                and            -   —OP(O)(OR¹¹)₂;        -   each R¹⁰ is independently selected from aryl,            C₃-C₈cycloalkyl, C₃-C₈heterocycloalkyl and heteroaryl,            wherein the aryl, C₃-C₈cycloalkyl, C₃-C₈heterocycloalkyl and            heteroaryl groups are optionally substituted with 1 to 3            substituents selected from halogen, —R⁸, —OR⁸, -LR⁹, -LOR⁹,            —N(R⁹)₂, —NR⁹C(O)R⁸,            -   —NR⁹CO₂R⁸, —CO₂R⁸, —C(O)R⁸ and —C(O)N(R⁹)₂;        -   R¹¹ and R¹² are independently selected from H, C₁-C₆alkyl,            C₁-C₆heteroalkyl, C₁-C₆haloalkyl, aryl, heteroaryl,            C₃-C₈cycloalkyl, and C₃-C₈heterocycloalkyl, wherein the            C₁-C₆alkyl, C₁-C₆heteroalkyl, C₁-C₆haloalkyl, aryl,            heteroaryl, C₃-C₈cycloalkyl, and C₃-C₈heterocycloalkyl            groups of R¹¹ and R¹² are each optionally substituted with 1            to 3 substituents independently selected from halogen, —CN,            R⁸, —OR⁸, —C(O)R⁸,            -   ⁻OC(O)R⁸, —C(O)OR⁸, —N(R⁹)₂, —NR⁸C(O)R⁸, —NR⁸C(O)OR⁸,                —C(O)N(R⁹)₂, C₃-C₈heterocycloalkyl, —S(O)₂R⁸,                —S(O)₂N(R⁹)₂, —NR⁹S(O)₂R⁸, C₁-C₆haloalkyl and                C₁-C₆haloalkoxy;        -   or R¹¹ and R¹² are each independently C₁-C₆alkyl and taken            together with the N atom to which they are attached form an            optionally substituted C₃-C₈heterocycloalkyl ring optionally            containing an additional heteroatom selected from N, O and            S;        -   each R¹³ is independently selected from halogen, —CN, -LR⁹,            -LOR⁹, —OLR⁹, -LR¹⁰, -LOR¹⁰, —OLR¹⁰, -LR⁸, -LOR⁸, —OLR⁸,            -LSR⁸, -LSR¹⁰, -LC(O)R⁸, —OLC(O)R⁸, -LC(O)OR⁸, -LC(O)R¹⁰,            -LOC(O)OR⁸, -LC(O)NR⁹R¹¹, -LC(O)NR⁹R⁸, -LN(R⁹)₂, -LNR⁹R⁸,            -LNR⁹R¹⁰, -LC(O)N(R⁹)₂, -LS(O)₂R⁸, -LS(O)R⁸, -LC(O)NR⁸OH,            -LNR⁹C(O)R⁸, -LNR⁹C(O)OR⁸, -LS(O)₂N(R⁹)₂, —OLS(O)₂N(R⁹)₂,            -LNR⁹S(O)₂R⁸, -LC(O)NR⁹LN(R⁹)₂, -LP(O)(OR⁸)₂, -LOP(O)(OR⁸)₂,            -LP(O)(OR¹⁰)₂ and -OLP(O)(OR¹⁰)₂;        -   each R^(A) is independently selected from —R⁸, —R⁷, —OR⁷,            —OR⁸, —R¹⁰, —OR¹⁰, —SR⁸, —NO₂, —CN, —N(R⁹)₂, —NR⁹C(O)R⁸,            —NR⁹C(S)R⁸, —NR⁹C(O)N(R⁹)₂, —NR⁹C(S)N(R⁹)₂, —NR⁹CO₂R⁸,            —NR⁹NR⁹C(O)R⁸, —NR⁹NR⁹C(O)N(R⁹)₂, —NR⁹NR⁹CO₂R⁸, —C(O)C(O)R⁸,            —C(O)CH₂C(O)R⁸, —CO₂R⁸, —(CH₂)_(n)CO₂R⁸, —C(O)R⁸, —C(S)R⁸,            —C(O)N(R⁹)₂, —C(S)N(R⁹)₂, —OC(O)N(R⁹)₂, —OC(O)R⁸,            —C(O)N(OR⁸)R⁸, —C(NOR⁸)R⁸, —S(O)₂R⁸, —S(O)₃R⁸, —SO₂N(R⁹)₂,            —S(O)R⁸, —NR⁹SO₂N(R⁹)₂, —NR⁹SO₂R⁸, —P(O)(OR⁸)₂,            —OP(O)(OR⁸)₂, —P(O)(OR¹⁰)₂, —OP(O)(OR¹⁰)₂, —N(OR⁸)R⁸,            —CH═CHCO₂R⁸, —C(═NH)—N(R⁹)₂, and —(CH₂)_(n)NHC(O)R⁸;        -   n is, independently at each occurrence, 0, 1, 2, 3, 4, 5, 6,            7 or 8;        -   each m is independently selected from 1, 2, 3, 4, 5 and 6,            and    -   t is 1, 2, 3, 4, 5, 6, 7 or 8; or (c) a pharmaceutically        acceptable salt of any of (a) or (b). Other benzonaphthyridine        compounds, and methods of making benzonaphthyridine compounds,        are described in WO 2009/111337. A benzonaphthyridine compound,        or a salt thereof, can be used on its own, or in combination        with one or more further compounds. For example, a        benzonaphthyridine compound can be used in combination with an        oil-in-water emulsion or a mineral-containing composition. In a        particular embodiment, a benzonaphthyridine compound is used in        combination with an oil-in-water emulsion (e.g. a squalene-water        emulsion, such as MF59) or a mineral-containing composition        (e.g., a mineral sald such as an aluminum salt or a calcium        salt).    -   A thiosemicarbazone compound, such as those disclosed in        reference 94. Methods of formulating, manufacturing, and        screening for active compounds are also described in        reference 94. The thiosemicarbazones are particularly effective        in the stimulation of human peripheral blood mononuclear cells        for the production of cytokines, such as TNF-α.    -   A tryptanthrin compound, such as those disclosed in        reference 95. Methods of formulating, manufacturing, and        screening for active compounds are also described in        reference 95. The thiosemicarbazones are particularly effective        in the stimulation of human peripheral blood mononuclear cells        for the production of cytokines, such as TNF-α.    -   A nucleoside analog, such as: (a) Isatorabine (ANA-245;        7-thia-8-oxoguanosine):

-   -   and prodrugs thereof; (b) ANA975; (c) ANA-025-1; (d) ANA380; (e)        the compounds disclosed in references 96 to 98; (f) a compound        having the formula:

-   -   -   wherein:        -   R₁ and R₂ are each independently H, halo, —NR_(a)R_(b), —OH,            C₁₋₆ alkoxy, substituted C₁₋₆ alkoxy, heterocyclyl,            substituted heterocyclyl, C₆₋₁₀ aryl, substituted C₆₋₁₀            aryl, C₁₋₆ alkyl, or substituted C₁₋₆ alkyl;        -   R₃ is absent, H, C₁₋₆ alkyl, substituted C₁₋₆ alkyl, C₆₋₁₀            aryl, substituted C₆₋₁₀ aryl, heterocyclyl, or substituted            heterocyclyl;        -   R₄ and R₅ are each independently H, halo, heterocyclyl,            substituted heterocyclyl, —C(O)—R_(d), C₁₋₆ alkyl,            substituted C₁₋₆ alkyl, or bound together to form a 5            membered ring as in R₄₋₅:

-   -   -   -   the binding being achieved at the bonds indicated by a

        -   X₁ and X₂ are each independently N, C, O, or S;

        -   R₈ is H, halo, —OH, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl,            —OH, —NR_(a)R_(b), —(CH₂)_(n)—O—R_(c), —O—(C₁₋₆ alkyl),            —S(O)_(p)R_(e), or —C(O)—R_(d);

        -   R₉ is H, C₁₋₆ alkyl, substituted C₁₋₆ alkyl, heterocyclyl,            substituted heterocyclyl or R_(9a), wherein R_(9a) is:

-   -   -   -   the binding being achieved at the bond indicated by a

        -   R₁₀ and R₁₁ are each independently H, halo, C₁₋₆ alkoxy,            substituted C₁₋₆ alkoxy, —NR_(a)R_(b), or —OH;

        -   each R_(a) and R_(b) is independently H, C₁₋₆ alkyl,            substituted C₁₋₆ alkyl, —C(O)R_(d), C₆₋₁₀ aryl;

        -   each R_(c) is independently H, phosphate, diphosphate,            triphosphate, C₁₋₆ alkyl, or substituted C₁₋₆ alkyl;

        -   each R_(d) is independently H, halo, C₁₋₆ alkyl, substituted            C₁₋₆ alkyl, C₁₋₆ alkoxy, substituted C₁₋₆ alkoxy, —NH₂,            —NH(C₁₋₆ alkyl), —NH(substituted C₁₋₆ alkyl), —N(C₁₋₆            alkyl)₂, —N(substituted C₁₋₆ alkyl)₂, C₆₋₁₀ aryl, or            heterocyclyl;

        -   each R_(e) is independently H, C₁₋₆ alkyl, substituted C₁₋₆            alkyl, C₆₋₁₀ aryl, substituted C₆₋₁₀ aryl, heterocyclyl, or            substituted heterocyclyl;

        -   each R_(f) is independently H, C₁₋₆ alkyl, substituted C₁₋₆            alkyl, —C(O)R_(d), phosphate, diphosphate, or triphosphate;

        -   each n is independently 0, 1, 2, or 3;

        -   each p is independently 0, 1, or 2; or

    -   or (g) a pharmaceutically acceptable salt of any of (a) to (f),        a tautomer of any of (a) to (f), or a pharmaceutically        acceptable salt of the tautomer.

    -   Loxoribine (7-allyl-8-oxoguanosine) (99).

    -   Compounds disclosed in reference 100, including: Acylpiperazine        compounds, Indoledione compounds, Tetrahydraisoquinoline (THIQ)        compounds, Benzocyclodione compounds, Aminoazavinyl compounds,        Aminobenzimidazole quinolinone (ABIQ) compounds (101, 102),        Hydrapthalamide compounds, Benzophenone compounds, Isoxazole        compounds, Sterol compounds, Quinazilinone compounds, Pyrrole        compounds (103), Anthraquinone compounds, Quinoxaline compounds,        Triazine compounds, Pyrazalopyrimidine compounds, and Benzazole        compounds (104).

    -   Compounds disclosed in reference 105.

    -   An aminoalkyl glucosaminide phosphate derivative, such as RC-529        (106, 107).

    -   A phosphazene, such as poly[di(carboxylatophenoxy)phosphazene]        (“PCPP”) as described, for example, in references 108 and 109.

    -   Small molecule immunopotentiators (SMIPs) such as:        -   N2-methyl-1-(2-methylpropyl)-1H-imidazo[4,5-c]quinoline-2,4-diamine        -   N2,N2-dimethyl-1-(2-methylpropyl)-1H-imidazo[4,5-c]quinoline-2,4-diamine        -   N2-ethyl-N2-methyl-1-(2-methylpropyl)-1H-imidazo[4,5-c]quinoline-2,4-diamine        -   N2-methyl-1-(2-methylpropyl)-N2-propyl-1H-imidazo[4,5-c]quinoline-2,4-diamine        -   1-(2-methylpropyl)-N2-propyl-TH-imidazo[4,5-c]quinoline-2,4-diamine        -   N2-butyl-1-(2-methylpropyl)-1H-imidazo[4,5-c]quinoline-2,4-diamine        -   N2-butyl-N2-methyl-1-(2-methylpropyl)-1H-imidazo[4,5-c]quinoline-2,4-diamine        -   N2-methyl-1-(2-methylpropyl)-N2-pentyl-1H-imidazo[4,5-c]quinoline-2,4-diamine        -   N2-methyl-1-(2-methylpropyl)-N2-prop-2-enyl-1H-imidazo[4,5-c]quinoline-2,4-diamine        -   1-(2-methylpropyl)-2-[(phenylmethyl)thio]-1H-imidazo[4,5-c]quinolin-4-amine        -   1-(2-methylpropyl)-2-(propylthio)-1H-imidazo[4,5-c]quinolin-4-amine        -   2-[[4-amino-1-(2-methylpropyl)-1H-imidazo[4,5-c]quinolin-2-yl](methyl)amino]ethanol        -   2-[[4-amino-1-(2-methylpropyl)-1H-imidazo[4,5-c]quinolin-2-yl](methyl)amino]ethyl            acetate        -   4-amino-1-(2-methylpropyl)-1,3-dihydro-2H-imidazo[4,5-c]quinolin-2-one        -   N2-butyl-1-(2-methylpropyl)-N4,N4-bis(phenylmethyl)-1H-imidazo[4,5-c]quinoline-2,4-diamine        -   N2-butyl-N2-methyl-1-(2-methylpropyl)-N4,N4-bis(phenylmethyl)-TH-imidazo[4,5-c]quinoline-2,4-diamine        -   N2-methyl-1-(2-methylpropyl)-N4,N4-bis(phenylmethyl)-1H-imidazo[4,5-c]quinoline-2,4-diamine        -   N2,N2-dimethyl-1-(2-methylpropyl)-N4,N4-bis(phenylmethyl)-1H-imidazo[4,5-c]quinoline-2,4-diamine        -   1-[4-amino-2-[methyl(propyl)amino]-1H-imidazo[4,5-c]quinolin-1-yl]-2-methylpropan-2-ol        -   1-[4-amino-2-(propylamino)-1H-imidazo[4,5-c]quinolin-1-yl]-2-methylpropan-2-ol        -   N4,N4-dibenzyl-1-(2-methoxy-2-methylpropyl)-N2-propyl-1H-imidazo[4,5-c]quinoline-2,4-diamine.

The cytokine-inducing agents for use in the present invention may bemodulators and/or agonists of Toll-Like Receptors (TLR). For example,they may be agonists of one or more of the human TLR1, TLR2, TLR3, TLR4,TLR7, TLR8, and/or TLR9 proteins. Preferred agents are agonists of TLR4(e.g., modified natural lipid As derived from enterobacteriallipopolysaccharides, phospholipid compounds, such as the syntheticphospholipid dimer, E6020), TLR7 (e.g., benzonaphthyridines,imidazoquinolines) and/or TLR9 (e.g., CpG oligonucleotides). Theseagents are useful for activating innate immunity pathways.

The cytokine-inducing agent can be added to the composition at variousstages during its production. For example, it may be within an antigencomposition, and this mixture can then be added to an oil-in-wateremulsion. As an alternative, it may be within an oil-in-water emulsion,in which case the agent can either be added to the emulsion componentsbefore emulsification, or it can be added to the emulsion afteremulsification. Similarly, the agent may be coacervated within theemulsion droplets. The location and distribution of thecytokine-inducing agent within the final composition will depend on itshydrophilic/lipophilic properties, e.g., the agent can be located in theaqueous phase, in the oil phase, and/or at the oil-water interface.

The cytokine-inducing agent can be conjugated to a separate agent, suchas an antigen (e.g., CRM197). A general review of conjugation techniquesfor small molecules is provided in ref. 110. As an alternative, theadjuvants may be non-covalently associated with additional agents, suchas by way of hydrophobic or ionic interactions.

Preferred cytokine-inducing agents are (a) benzonapthridine compounds;(b) immunostimulatory oligonucleotides and (c) 3dMPL.

Immunostimulatory oligonucleotides can include nucleotidemodifications/analogs such as phosphorothioate modifications and can bedouble-stranded or (except for RNA) single-stranded. References 111,112, and 113disclose possible analog substitutions, e.g., replacement ofguanosine with 2′-deoxy-7-deazaguanosine. The adjuvant effect of CpGoligonucleotides is further discussed in refs. 114 to 119. A CpGsequence may be directed to TLR9, such as the motif GTCGTT or TTCGTT(120). The CpG sequence may be specific for inducing a Th1 immuneresponse, such as a CpG-A ODN (oligodeoxynucleotide), or it may be morespecific for inducing a B cell response, such a CpG-B ODN. CpG-A andCpG-B ODNs are discussed in refs. 121-123. Preferably, the CpG is aCpG-A ODN. Preferably, the CpG oligonucleotide is constructed so thatthe 5′ end is accessible for receptor recognition. Optionally, two CpGoligonucleotide sequences may be attached at their 3′ ends to form“immunomers”. See, for example, references 120 & 124-126. A useful CpGadjuvant is CpG7909, also known as PROMUNE™ (Coley Pharmaceutical Group,Inc.).

As an alternative, or in addition, to using CpG sequences, TpG sequencescan be used (127). These oligonucleotides may be free from unmethylatedCpG motifs.

The immunostimulatory oligonucleotide may be pyrimidine-rich. Forexample, it may comprise more than one consecutive thymidine nucleotide(e.g., TTTT, as disclosed in ref. 127), and/or it may have a nucleotidecomposition with >25% thymidine (e.g., >35%, >40%, >50%, >60%, >80%,etc.). For example, it may comprise more than one consecutive cytosinenucleotide (e.g., CCCC, as disclosed in ref. 127), and/or it may have anucleotide composition with >25% cytosine(e.g., >35%, >40%, >50%, >60%, >80%, etc.). These oligonucleotides maybe free from unmethylated CpG motifs.

Immunostimulatory oligonucleotides will typically comprise at least 20nucleotides. They may comprise fewer than 100 nucleotides.

3dMPL (also known as 3 de-O-acylated monophosphoryl lipid A or3-O-desacyl-4′-monophosphoryl lipid A) is an adjuvant in which position3 of the reducing end glucosamine in monophosphoryl lipid A has beende-acylated. 3dMPL has been prepared from a heptoseless mutant ofSalmonella minnesota, and is chemically similar to lipid A but lacks anacid-labile phosphoryl group and a base-labile acyl group. It activatescells of the monocyte/macrophage lineage and stimulates release ofseveral cytokines, including IL-1, IL-12, TNF-α and GM-CSF (see alsoref. 128). Preparation of 3dMPL was originally described in reference129.

3dMPL can take the form of a mixture of related molecules, varying bytheir acylation (e.g., having 3, 4, 5 or 6 acyl chains, which may be ofdifferent lengths). The two glucosamine (also known as2-deoxy-2-amino-glucose) monosaccharides are N-acylated at their2-position carbons (i.e., at positions 2 and 2′), and there is alsoO-acylation at the 3′ position. The group attached to carbon 2 hasformula —NH—CO—CH₂—CR¹R^(1′). The group attached to carbon 2′ hasformula —NH—CO—CH₂—CR²R^(2′). The group attached to carbon 3′ hasformula —O—CO—CH₂—CR³R^(3′). A representative structure is:

Groups R¹, R² and R³ are each independently —(CH₂)_(n)—CH₃. The value ofn is preferably between 8 and 16, more preferably between 9 and 12, andis most preferably 10.

Groups R^(1′), R^(2′) and R^(3′) can each independently be: (a) —H; (b)—OH; or (c) —O—CO—R⁴, where R⁴ is either —H or —(CH₂)_(m)CH₃, whereinthe value of m is preferably between 8 and 16, and is more preferably10, 12 or 14. At the 2 position, m is preferably 14. At the 2′ position,m is preferably 10. At the 3′ position, m is preferably 12. GroupsR^(1′), R^(2′) and R^(3′) are thus preferably —O-acyl groups fromdodecanoic acid, tetradecanoic acid or hexadecanoic acid.

When all of R^(1′), R^(2′) and R^(3′) are —H then the 3dMPL has only 3acyl chains (one on each of positions 2, 2′ and 3′). When only two ofR^(1′), R^(2′) and R^(3′) are —H then the 3dMPL can have 4 acyl chains.When only one of R^(1′), R^(2′) and R^(3′) is —H then the 3dMPL can have5 acyl chains. When none of R^(1′), R^(2′) and R^(3′) is —H then the3dMPL can have 6 acyl chains. The 3dMPL adjuvant used according to theinvention can be a mixture of these forms, with from 3 to 6 acyl chains,but it is preferred to include 3dMPL with 6 acyl chains in the mixture,and in particular to ensure that the hexaacyl chain form makes up atleast 10% by weight of the total 3dMPL e.g., >20%, >30%, >40%, >50% ormore. 3dMPL with 6 acyl chains has been found to be the mostadjuvant-active form.

Thus the most preferred form of 3dMPL for inclusion in compositions ofthe invention has formula (IV), shown below.

Where 3dMPL is used in the form of a mixture then references to amountsor concentrations of 3dMPL in compositions of the invention refer to thecombined 3dMPL species in the mixture.

In aqueous conditions, 3dMPL can form micellar aggregates or particleswith different sizes e.g., with a diameter <150 nm or >500 nm. Either orboth of these can be used with the invention, and the better particlescan be selected by routine assay. Smaller particles (e.g., small enoughto give a clear aqueous suspension of 3dMPL) are preferred for useaccording to the invention because of their superior activity (130).Preferred particles have a mean diameter less than 220 nm, morepreferably less than 200 nm or less than 150 nm or less than 120 nm, andcan even have a mean diameter less than 100 nm. In most cases, however,the mean diameter will not be lower than 50 nm. These particles aresmall enough to be suitable for filter sterilization. Particle diametercan be assessed by the routine technique of dynamic light scattering,which reveals a mean particle diameter. Where a particle is said to havea diameter of x nm, there will generally be a distribution of particlesabout this mean, but at least 50% by number(e.g., >60%, >70%, >80%, >90%, or more) of the particles will have adiameter within the range x+25%.

3dMPL can advantageously be used in combination with an oil-in-wateremulsion. Substantially all of the 3dMPL may be located in the aqueousphase of the emulsion.

The 3dMPL can be used on its own, or in combination with one or morefurther compounds. For example, it is known to use 3dMPL in combinationwith the QS21 saponin (131) (including in an oil-in-water emulsion(132)), with an immunostimulatory oligonucleotide, with both QS21 and animmunostimulatory oligonucleotide, with aluminum phosphate (133), withaluminum hydroxide (134), or with both aluminum phosphate and aluminumhydroxide.

Fatty Adjuvants

Fatty adjuvants that can be used with the invention include theoil-in-water emulsions described above, and also include, for example:

-   -   A phospholipid compound of formula I, II or III, or a salt        thereof:

as defined in reference 135, such as ‘ER 803058’, ‘ER 803732’, ‘ER804053’, ER 804058’, ‘ER 804059’, ‘ER 804442’, ‘ER 804680’, ‘ER 804764’,ER 803022 or ‘ER 804057’ e.g.:

-   -   ER804057 is also called E6020. A phospholipid compound of        formula I, II or III, or a salt thereof, can be used on its own,        or in combination with one or more further compounds. For        example, a compound of formula I, II or III, can be used in        combination with an oil-in-water emulsion or a        mineral-containing composition. In a particular embodiment,        E6020 is used in combination with an oil-in-water emulsion (e.g.        a squalene-water emulsion, such as MF59) or a mineral-containing        composition (e.g., a mineral sald such as an aluminum salt or a        calcium salt).    -   Derivatives of lipid A from Escherichia coli such as OM-174        (described in refs. 136 & 137).    -   A formulation of a cationic lipid and a (usually neutral)        co-lipid, such as        aminopropyl-dimethyl-myristoleyloxy-propanaminium        bromide-diphytanoylphosphatidyl-ethanolamine (“VAXFECTIN™”) or        aminopropyl-dimethyl-bis-dodecyloxy-propanaminium        bromide-dioleoylphosphatidyl-ethanolamine (“GAP-DLRIE:DOPE”).        Formulations containing        (+)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(syn-9-tetradeceneyloxy)-1-propanaminium        salts are preferred (138).    -   3-O-deacylated monophosphoryl lipid A (see above).    -   Compounds containing lipids linked to a phosphate-containing        acyclic backbone, such as the TLR4 antagonist E5564 (139, 140):

-   -   Lipopeptides (i.e., compounds comprising one or more fatty acid        residues and two or more amino acid residues), such as        lipopeptides based on glycerylcysteine. Specific examples of        such peptides include compounds of the following formula

in which each of R¹ and R² represents a saturated or unsaturated,aliphatic or mixed aliphatic-cycloaliphatic hydrocarbon radical havingfrom 8 to 30, preferably 11 to 21, carbon atoms that is optionally alsosubstituted by oxygen functions, R³ represents hydrogen or the radicalR₁—CO—O—CH₂— in which R¹ has the same meaning as above, and X representsan amino acid bonded by a peptide linkage and having a free, esterifiedor amidated carboxy group, or an amino acid sequence of from 2 to 10amino acids of which the terminal carboxy group is in free, esterifiedor amidated form. In certain embodiments, the amino acid sequencecomprises a D-amino acid, for example, D-glutamic acid (D-Glu) orD-gamma-carboxy-glutamic acid (D-Gla).

Bacterial lipopeptides generally recognize TLR2, without requiring TLR6to participate. (TLRs operate cooperatively to provide specificrecognition of various triggers, and TLR2 plus TLR6 together recognizepeptidoglycans, while TLR2 recognizes lipopeptides without TLR6.) Theseare sometimes classified as natural lipopeptides and syntheticlipopeptides. Synthetic lipopeptides tend to behave similarly, and areprimarily recognized by TLR2.

Lipopeptides suitable for use as adjuvants include compounds have theformula:

where the chiral center labeled * and the one labeled *** are both inthe R configuration; the chiral center labeled ** is either in the R orS configuration;each R^(1a) and R^(1b) is independently an aliphatic orcycloaliphatic-aliphatic hydrocarbon group having 7-21 carbon atoms,optionally substituted by oxygen functions, or one of Ria and R^(1b),but not both, is H;

-   R² is an aliphatic or cycloaliphatic hydrocarbon group having 1-21    carbon atoms and optionally substituted by oxygen functions;-   n is 0 or 1;-   As represents either —O-Kw-CO— or —NH-Kw-CO—, where Kw is an    aliphatic hydrocarbon group having 1-12 carbon atoms;-   As¹ is a D- or L-alpha-amino acid;-   Z¹ and Z² each independently represent —OH or the N-terminal radical    of a D- or L-alpha amino acid of an amino-(lower alkane)-sulfonic    acid or of a peptide having up to 6 amino acids selected from the D-    and L-alpha aminocarboxylic acids and amino-lower alkyl-sulfonic    acids; and-   Z³ is H or —CO—Z⁴, where Z⁴ is —OH or the N-terminal radical of a D-    or L-alpha amino acid of an amino-(lower alkane)-sulfonic acid or of    a peptide having up to 6 amino acids selected from the D and L-alpha    aminocarboxylic acids and amino-lower alkyl-sulfonic acids; or an    ester or amide formed from the carboxylic acid of such compounds.    Suitable amides include —NH₂ and NH(lower alkyl), and suitable    esters include C₁-C₄ alkyl esters.-   (lower alkyl or lower alkane, as used herein, refers to C₁-C₆    straight chain or branched alkyls).

Such compounds are described in more detail in U.S. Pat. No. 4,666,886.In one particular embodiment, the lipopeptide has the formula:

Another example of a lipopeptide species is called LP40, and is anagonist of TLR2. Akdis, et al., Eur. J. Immunology, 33: 2717-26 (2003).

These are related to a known class of lipopeptides from E. coli,referred to as murein lipoproteins. Certain partial degradation productsof those proteins called murein lipopetides are described in Hantke, etal., Eur. J Biochem., 34: 284-296 (1973). These comprise a peptidelinked to N-acetyl muramic acid and are thus related to Muramylpeptides, which are described in Baschang, et al., Tetrahedron, 45(20):6331-6360 (1989).

Aluminum Salt Adjuvants

The adjuvants known as “aluminum hydroxide” and “aluminum phosphate” maybe used. These names are conventional, but are used for convenienceonly, as neither is a precise description of the actual chemicalcompound which is present (e.g., see chapter 9 of reference 74). Theinvention can use any of the “hydroxide” or “phosphate” adjuvants thatare in general use as adjuvants.

The adjuvants known as “aluminum hydroxide” are typically aluminumoxyhydroxide salts, which are usually at least partially crystalline.Aluminum oxyhydroxide, which can be represented by the formula AlO(OH),can be distinguished from other aluminum compounds, such as aluminumhydroxide Al(OH)₃, by infrared (IR) spectroscopy, in particular by thepresence of an adsorption band at 1070 cm⁻¹ and a strong shoulder at3090-3100 cm⁻¹ (chapter 9 of ref. 74). The degree of crystallinity of analuminum hydroxide adjuvant is reflected by the width of the diffractionband at half height (WHH), with poorly-crystalline particles showinggreater line broadening due to smaller crystallite sizes. The surfacearea increases as WHH increases, and adjuvants with higher WHH valueshave been seen to have greater capacity for antigen adsorption. Afibrous morphology (e.g., as seen in transmission electron micrographs)is typical for aluminum hydroxide adjuvants. The pI of aluminumhydroxide adjuvants is typically about 11, i.e., the adjuvant itself hasa positive surface charge at physiological pH. Adsorptive capacities ofbetween 1.8-2.6 mg protein per mg Al⁺⁺⁺ at pH 7.4 have been reported foraluminum hydroxide adjuvants.

The adjuvants known as “aluminum phosphate” are typically aluminumhydroxyphosphates, often also containing a small amount of sulfate(i.e., aluminum hydroxyphosphate sulfate). They may be obtained byprecipitation, and the reaction conditions and concentrations duringprecipitation influence the degree of substitution of phosphate forhydroxyl in the salt. Hydroxyphosphates generally have a PO₄/Al molarratio between 0.3 and 1.2. Hydroxyphosphates can be distinguished fromstrict AlPO₄ by the presence of hydroxyl groups. For example, an IRspectrum band at 3164 cm⁻¹ (e.g., when heated to 200° C.) indicates thepresence of structural hydroxyls (ch. 9 of ref. 74)

The PO₄/Al³⁺ molar ratio of an aluminum phosphate adjuvant willgenerally be between 0.3 and 1.2, preferably between 0.8 and 1.2, andmore preferably 0.95+0.1. The aluminum phosphate will generally beamorphous, particularly for hydroxyphosphate salts. A typical adjuvantis amorphous aluminum hydroxyphosphate with PO₄/Al molar ratio between0.84 and 0.92, included at 0.6 mg Al³⁺/ml. The aluminum phosphate willgenerally be particulate (e.g., plate-like morphology as seen intransmission electron micrographs). Typical diameters of the particlesare in the range 0.5-20 μm (e.g., about 5-10 μm) after any antigenadsorption. Adsorptive capacities of between 0.7-1.5 mg protein per mgAl⁺⁺⁺ at pH 7.4 have been reported for aluminum phosphate adjuvants.

The point of zero charge (PZC) of aluminum phosphate is inverselyrelated to the degree of substitution of phosphate for hydroxyl, andthis degree of substitution can vary depending on reaction conditionsand concentration of reactants used for preparing the salt byprecipitation. PZC is also altered by changing the concentration of freephosphate ions in solution (more phosphate=more acidic PZC) or by addinga buffer such as a histidine buffer (makes PZC more basic). Aluminumphosphates used according to the invention will generally have a PZC ofbetween 4.0 and 7.0, more preferably between 5.0 and 6.5, e.g., about5.7.

Suspensions of aluminum salts used to prepare compositions of theinvention may contain a buffer (e.g., a phosphate or a histidine or aTris buffer), but this is not always necessary. The suspensions arepreferably sterile and pyrogen-free. A suspension may include freeaqueous phosphate ions e.g., present at a concentration between 1.0 and20 mM, preferably between 5 and 15 mM, and more preferably about 10 mM.The suspensions may also comprise sodium chloride.

The invention can use a mixture of both an aluminum hydroxide and analuminum phosphate. In this case there may be more aluminum phosphatethan hydroxide e.g., a weight ratio of at least 2:1e.g., >5:1, >6:1, >7:1, >8:1, >9:1, etc.

The concentration of Al⁺⁺⁺ in a composition for administration to apatient is preferably less than 10 mg/ml e.g., <5 mg/ml, <4 mg/ml, <3mg/ml, <2 mg/ml, <1 mg/ml, etc. A preferred range is between 0.3 and 1mg/ml. A maximum of 0.85 mg/dose is preferred.

As well as including one or more aluminum salt adjuvants, the adjuvantcomponent may include one or more further adjuvant or immunostimulatingagents. Such additional components include, but are not limited to: abenzonaphthyridine compound, a 3-O-deacylated monophosphoryl lipid Aadjuvant (‘3d-MPL’); and/or an oil-in-water emulsion. 3d-MPL has alsobeen referred to as 3 de-O-acylated monophosphoryl lipid A or as3-O-desacyl-4′-monophosphoryl lipid A. The name indicates that position3 of the reducing end glucosamine in monophosphoryl lipid A isde-acylated. It has been prepared from a heptoseless mutant of S.minnesota, and is chemically similar to lipid A but lacks an acid-labilephosphoryl group and a base-labile acyl group. It activates cells of themonocyte/macrophage lineage and stimulates release of several cytokines,including IL-1, IL-12, TNF-α and GM-CSF. Preparation of 3d-MPL wasoriginally described in reference 129, and the product has beenmanufactured and sold by Corixa Corporation under the name MPL™. Furtherdetails can be found in refs 82 to 85.

The use of an aluminum hydroxide and/or aluminum phosphate adjuvant isuseful, particularly in children, and antigens are generally adsorbed tothese salts. Squalene-in-water emulsions are also preferred,particularly in the elderly. Useful adjuvant combinations includecombinations of Th1 and Th2 adjuvants such as CpG and alum, orresiquimod and alum. A combination of aluminum phosphate and 3dMPL maybe used. Other combinations that may be used include: alum and abenzonapthridine compound or a SMIP, a squalene-in-water emulsion (suchas MF59) and a benzonapthridine compound or a SMIP, and E6020 and asqualene-in-water emulsion, such as MF59) or alum.

The compositions of the invention may elicit both a cell mediated immuneresponse as well as a humoral immune response.

Two types of T cells, CD4 and CD8 cells, are generally thought necessaryto initiate and/or enhance cell mediated immunity and humoral immunity.CD8 T cells can express a CD8 co-receptor and are commonly referred toas Cytotoxic T lymphocytes (CTLs). CD8 T cells are able to recognized orinteract with antigens displayed on MHC Class I molecules.

CD4 T cells can express a CD4 co-receptor and are commonly referred toas T helper cells. CD4 T cells are able to recognize antigenic peptidesbound to MHC class II molecules. Upon interaction with a MHC class IImolecule, the CD4 cells can secrete factors such as cytokines. Thesesecreted cytokines can activate B cells, cytotoxic T cells, macrophages,and other cells that participate in an immune response. Helper T cellsor CD4+ cells can be further divided into two functionally distinctsubsets: TH1 phenotype and TH2 phenotypes which differ in their cytokineand effector function.

Activated TH1 cells enhance cellular immunity (including an increase inantigen-specific CTL production) and are therefore of particular valuein responding to intracellular infections. Activated TH1 cells maysecrete one or more of IL-2, IFN-γ, and TNF-β. A TH1 immune response mayresult in local inflammatory reactions by activating macrophages, NK(natural killer) cells, and CD8 cytotoxic T cells (CTLs). A TH1 immuneresponse may also act to expand the immune response by stimulatinggrowth of B and T cells with IL-12. TH1 stimulated B cells may secreteIgG2a.

Activated TH2 cells enhance antibody production and are therefore ofvalue in responding to extracellular infections. Activated TH2 cells maysecrete one or more of IL-4, IL-5, IL-6, and IL-10. A TH2 immuneresponse may result in the production of IgG1, IgE, IgA and memory Bcells for future protection.

An enhanced immune response may include one or more of an enhanced TH1immune response and a TH2 immune response.

A TH1 immune response may include one or more of an increase in CTLs, anincrease in one or more of the cytokines associated with a TH1 immuneresponse (such as IL-2, IFN-γ, and TNF-β), an increase in activatedmacrophages, an increase in NK activity, or an increase in theproduction of IgG2a. Preferably, the enhanced TH1 immune response willinclude an increase in IgG2a production.

A TH1 immune response may be elicited using a TH1 adjuvant. A TH1adjuvant will generally elicit increased levels of IgG2a productionrelative to immunization of the antigen without adjuvant. TH1 adjuvantssuitable for use in the invention may include for example saponinformulations, virosomes and virus like particles, non-toxic derivativesof enterobacterial lipopolysaccharide (LPS), immunostimulatoryoligonucleotides. Immunostimulatory oligonucleotides, such asoligonucleotides containing a CpG motif, are preferred TH1 adjuvants foruse in the invention.

A TH2 immune response may include one or more of an increase in one ormore of the cytokines associated with a TH2 immune response (such asIL-4, IL-5, IL-6 and IL-10), or an increase in the production of IgG1,IgE, IgA and memory B cells. Preferably, the enhanced TH2 immuneresponse will include an increase in IgG1 production.

A TH2 immune response may be elicited using a TH2 adjuvant. A TH2adjuvant will generally elicit increased levels of IgG1 productionrelative to immunization of the antigen without adjuvant. TH2 adjuvantssuitable for use in the invention include, for example, mineralcontaining compositions, oil-emulsions, and ADP-ribosylating toxins anddetoxified derivatives thereof. Mineral containing compositions, such asaluminium salts are preferred TH2 adjuvants for use in the invention.

A composition may include a combination of a TH1 adjuvant and a TH2adjuvant. Preferably, such a composition elicits an enhanced TH1 and anenhanced TH2 response, i.e., an increase in the production of both IgG1and IgG2a production relative to immunization without an adjuvant. Stillmore preferably, the composition comprising a combination of a TH1 and aTH2 adjuvant elicits an increased TH1 and/or an increased TH2 immuneresponse relative to immunization with a single adjuvant (i.e., relativeto immunization with a TH1 adjuvant alone or immunization with a TH2adjuvant alone).

The immune response may be one or both of a TH1 immune response and aTH2 response. Preferably, immune response provides for one or both of anenhanced TH1 response and an enhanced TH2 response.

The enhanced immune response may be one or both of a systemic and amucosal immune response. Preferably, the immune response provides forone or both of an enhanced systemic and an enhanced mucosal immuneresponse. Preferably the mucosal immune response is a TH2 immuneresponse. Preferably, the mucosal immune response includes an increasein the production of IgA.

Methods of Treatment, and Administration

Compositions of the invention are suitable for administration tomammals, and the invention provides a method of inducing an immuneresponse in a mammal, comprising the step of administering a composition(e.g., an immunogenic composition) of the invention to the mammal. Thecompositions (e.g., an immunogenic composition) can be used to produce avaccine formulation for immunizing a mammal. The mammal is typically ahuman, and the RSV F protein ecto-domain is typically a human RSV Fprotein ecto-domain. However, the mammal can be any other mammal that issusceptible to infection with RSV, such as a cow that can be infectedwith bovine RSV. For example, the immune response may be raisedfollowing administration of a purified RSV F protein, an alphavirusparticle, or self-replicating RNA.

The invention also provides a composition of the invention for use as amedicament, e.g., for use in immunizing a patient against RSV infection.

The invention also provides the use of a polypeptide as described abovein the manufacture of a medicament for raising an immune response in apatient.

The immune response raised by these methods and uses will generallyinclude an antibody response, preferably a protective antibody response.Methods for assessing antibody responses after RSV vaccination are wellknown in the art.

Compositions of the invention can be administered in a number ofsuitable ways, such as intramuscular injection (e.g., into the arm orleg), subcutaneous injection, intranasal administration, oraladministration, intradermal administration, transcutaneousadministration, transdermal administration, and the like. Theappropriate route of administration will be dependent upon the age,health and other characteristics of the mammal. A clinician will be ableto determine an appropriate route of administration based on these andother factors.

Immunogenic compositions, and vaccine formulations, may be used to treatboth children and adults, including pregnant women. Thus a subject maybe less than 1 year old, 1-5 years old, 5-15 years old, 15-55 years old,or at least 55 years old. Preferred subjects for receiving the vaccinesare the elderly (e.g., >50 years old, >60 years old, and preferably >65years), the young (e.g., <6 years old, such as 4-6 years old, <5 yearsold), and pregnant women. The vaccines are not suitable solely for thesegroups, however, and may be used more generally in a population.

Treatment can be by a single dose schedule or a multiple dose schedule.Multiple doses may be used in a primary immunization schedule and/or ina booster immunization schedule. In a multiple dose schedule the variousdoses may be given by the same or different routes, e.g., a parenteralprime and mucosal boost, a mucosal prime and parenteral boost, etc.Administration of more than one dose (typically two doses) isparticularly useful in immunologically naive patients. Multiple doseswill typically be administered at least 1 week apart (e.g., about 2weeks, about 3 weeks, about 4 weeks, about 6 weeks, about 8 weeks, about10 weeks, about 12 weeks, about 16 weeks, and the like.). P

Vaccine formulations produced using a composition of the invention maybe administered to patients at substantially the same time as (e.g.,during the same medical consultation or visit to a healthcareprofessional or vaccination centre) other vaccines.

Further Aspects of the Invention

The invention also provides a polypeptide (e.g., recombinantpolypeptide) comprising a first domain and a second domain, wherein (i)the first domain comprises a RSV F glycoprotein ectodomain, in whole orpart, and (ii) the second domain comprises a heterologousoligomerization domain. Further details are provided above. If theoligomerization domain comprises a heptad sequence (e.g., the sequencefrom GCN described above) then it is preferably in heptad repeat phasewith the HR2 sequence (if present) of the ectodomain.

The invention also provides nucleic acid (e.g., DNA) encoding thispolypeptide. It also provides vectors including such nucleic acids, andhost cells including such vectors. The vectors may be used for, e.g.,recombinant expression purposes, nucleic acid immunization, etc.

The invention also provides a composition comprising moleculescomprising RSV F glycoprotein ectodomains, wherein the ectodomains of atleast 50% (e.g., 50%, 60%, 70%, 80%, 85%, 90%, 95% or 100%) of themolecules are present in a pre-fusion conformation.

Other Viruses

As well as being used with human RSV, the invention may be used withother members of the Pneumoviridae and Paramyxoviridae, including, butnot limited to, bovine respiratory syncytial virus, parainfluenzavirus1, parainflueznavirus 2, parainfluenzavirus 3, and parainfluenzavirus 5.

Thus the invention provides an immunogenic composition comprising a Fglycoprotein from a Pneumoviridae or Paramyxoviridae, wherein the Fglycoprotein is in pre-fusion conformation.

The invention also provides an immunogenic composition comprising apolypeptide that displays an epitope present in a pre-fusionconformation of the F glycoprotein of a Pneumoviridae orParamyxoviridae, but absent the glycoprotein's post fusion conformation.

The invention also provides a polypeptide comprising a first domain anda second domain, wherein (i) the first domain comprises an ectodomain ofthe F glycoprotein of a Pneumoviridae or Paramyxoviridae, in whole orpart, and (ii) the second domain comprises a heterologousoligomerization domain.

The invention also provides these polypeptides and compositions for usein immunization, etc.

The invention also provides a composition comprising moleculescomprising RSV F glycoprotein ectodomains, wherein the ectodomains of atleast 50% (e.g., 50%, 60%, 70%, 80%, 85%, 90%, 95% or 100%) of themolecules are present in a pre-fusion conformation or an intermediateconformation.

RSV F Protein Ecto-Domain Polypeptides

Particular RSV F protein ecto-domain polypeptides are used or includedin some embodiments of the invention. Some of the particular RSV Fprotein ecto-domain polypeptides contain altered amino acid sequencesfrom about position 100 to about position 161. The amino acid sequencesfrom position 100 to position 150 for several particular RSV F proteinecto-domain polypeptides are shown in FIG. 1C. Amino acid sequences ofseveral particular RSV F protein ecto-domain polypeptides are presentedherein, e.g., in Example 1.

General

The term “comprising” encompasses “including” as well as “consisting”and “consisting essentially of” e.g., a composition “comprising” X mayconsist exclusively of X or may include something additional e.g., X+Y.

The word “substantially” does not exclude “completely” e.g., acomposition which is “substantially free” from Y may be completely freefrom Y. Where necessary, the word “substantially” may be omitted fromthe definition of the invention.

The term “about” in relation to a numerical value x means, for example,x±10%.

Unless specifically stated, a process comprising a step of mixing two ormore components does not require any specific order of mixing. Thuscomponents can be mixed in any order. Where there are three componentsthen two components can be combined with each other, and then thecombination may be combined with the third component, etc.

Where animal (and particularly bovine) materials are used in the cultureof cells, they should be obtained from sources that are free fromtransmissible spongiform encaphalopathies (TSEs), and in particular freefrom bovine spongiform encephalopathy (BSE). Overall, it is preferred toculture cells in the total absence of animal-derived materials.

Where a compound is administered to the body as part of a compositionthen that compound may alternatively be replaced by a suitable prodrug.

Where a cell substrate is used for reassortment or reverse geneticsprocedures, it is preferably one that has been approved for use in humanvaccine production e.g., as in Ph Eur general chapter 5.2.3.

Identity between polypeptide sequences is preferably determined by theSmith-Waterman homology search algorithm as implemented in the MPSRCHprogram (Oxford Molecular), using an affine gap search with parametersgap open penalty=12 and gap extension penalty=1.

TABLE 1 Phospholipids DDPC1,2-Didecanoyl-sn-Glycero-3-phosphatidylcholine DEPA1,2-Dierucoyl-sn-Glycero-3-Phosphate DEPC1,2-Erucoyl-sn-Glycero-3-phosphatidylcholine DEPE1,2-Dierucoyl-sn-Glycero-3-phosphatidylethanolamine DEPG1,2-Dierucoyl-sn-Glycero-3[Phosphatidyl-rac-(1-glycerol...) DLOPC1,2-Linoleoyl-sn-Glycero-3-phosphatidylcholine DLPA1,2-Dilauroyl-sn-Glycero-3-Phosphate DLPC1,2-Dilauroyl-sn-Glycero-3-phosphatidylcholine DLPE1,2-Dilauroyl-sn-Glycero-3-phosphatidylethanolamine DLPG1,2-Dilauroyl-sn-Glycero-3 [Phosphatidyl-rac-(1-glycerol...) DLPS1,2-Dilauroyl-sn-Glycero-3-phosphatidylserine DMG1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine DMPA1,2-Dimyristoyl-sn-Glycero-3-Phosphate DMPC1,2-Dimyristoyl-sn-Glycero-3-phosphatidylcholine DMPE1,2-Dimyristoyl-sn-Glycero-3-phosphatidylethanolamine DMPG1,2-Myristoyl-sn-Glycero-3[Phosphatidyl-rac-(1-glycerol...) DMPS1,2-Dimyristoyl-sn-Glycero-3-phosphatidylserine DOPA1,2-Dioleoyl-sn-Glycero-3-Phosphate DOPC1,2-Dioleoyl-sn-Glycero-3-phosphatidylcholine DOPE1,2-Dioleoyl-sn-Glycero-3-phosphatidylethanolamine DOPG1,2-Dioleoyl-sn-Glycero-3[Phosphatidyl-rac-(1-glycerol...) DOPS1,2-Dioleoyl-sn-Glycero-3-phosphatidylserine DPPA1,2-Dipalmitoyl-sn-Glycero-3-Phosphate DPPC1,2-Dipalmitoyl-sn-Glycero-3-phosphatidylcholine DPPE1,2-Dipalmitoyl-sn-Glycero-3-phosphatidylethanolamine DPPG1,2-Dipalmitoyl-sn-Glycero-3[Phosphatidyl-rac-(1-glycerol...) DPPS1,2-Dipalmitoyl-sn-Glycero-3-phosphatidylserine DPyPE1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine DSPA1,2-Distearoyl-sn-Glycero-3-Phosphate DSPC1,2-Distearoyl-sn-Glycero-3-phosphatidylcholine DSPEl1,2-Diostearpyl-sn-Glycero-3-phosphatidylethanolamine DSPG1,2-Distearoyl-sn-Glycero-3[Phosphatidyl-rac-(1-glycerol...) DSPS1,2-Distearoyl-sn-Glycero-3-phosphatidylserine EPC Egg-PC HEPCHydrogenated Egg PC HSPC High purity Hydrogenated Soy PC HSPCHydrogenated Soy PC LYSOPC MYRISTIC1-Myristoyl-sn-Glycero-3-phosphatidylcholine LYSOPC PALMITIC1-Palmitoyl-sn-Glycero-3-phosphatidylcholine LYSOPC STEARIC1-Stearoyl-sn-Glycero-3-phosphatidylcholine Milk Sphingomyelin 1-Myristoyl,2-palmitoyl-sn-Glycero 3-phosphatidylcholine MPPC MSPC1-Myristoyl,2-stearoyl-sn-Glycero-3-phosphatidylcholine PMPC1-Palmitoyl,2-myristoyl-sn-Glycero-3-phosphatidylcholine POPC1-Palmitoyl,2-oleoyl-sn-Glycero-3-phosphatidylcholine POPE1-Palmitoyl-2-oleoyl-sn-Glycero-3-phosphatidylethanolamine POPG1,2-Dioleoyl-sn-Gly cero-3 [Phosphatidyl-rac-(1-glycerol)...] PSPC1-Palmitoyl,2-stearoyl-sn-Glycero-3-phosphatidylcholine SMPC1-Stearoyl,2-myristoyl-sn-Glycero-3-phosphatidylcholine SOPC1-Stearoyl,2-oleoyl-sn-Glycero-3-phosphatidylcholine SPPC1-Stearoyl,2-palmitoyl-sn-Glycero-3-phosphatidylcholine

EXEMPLIFICATION Example 1—RSV F Polypeptides

This example provides sequences of a number of examples of polypeptides(e.g., that contain signal sequences) and nucleic acid sequences thatmay be used to express RSV F polypeptides of the present invention. Thepresented amino acid sequences include the signal peptide and contain anoptional C-terminal linker and His tag (GGSAGSGHHHHHH (SEQ ID NO:90)).When these polypeptides are produced in host cells, the polypeptide willusually be processed by the cell to remove the signal peptide and, asdescribed herein, some of the polypeptides will be cleaved, for exampleat unmodified furin cleavage sites. The invention includes compositionsthat contain, all forms of the particular RSV F protein ecto-domainpolypeptides disclosed herein, including mature forms, which lack thesignal peptide, forms that may be cleaved into subunits that comprise F₁and F₂, and forms that lack the optional C-terminal His tag. Thefollowing examples are merely illustrative of the scope of the presentinvention and therefore are not intended to limit the scope in any way.

An example of wild-type furin cleavage is RSV F wild type Truncated HIS(SEQ ID NO:84).

Examples of polypeptides that can be produced as monomers include: RSV FFurx (SEQ ID NO:45); RSV F old furx Truncated HIS (SEQ ID NO:88); RSV FFurx R113Q K123N K124N Truncated HIS (SEQ ID NO:89); RSV F delp21 furxTruncated HIS (SEQ ID NO:47); and RSV F delP23 furx Truncated HIS (SEQID NO:48).

Examples of polypeptides that can be produced as trimers include: RSV FN-term Furin Truncated HIS (SEQ ID NO:85); RSV F Fusion DeletionTruncated HIS (SEQ ID NO:67); and RSV F Fusion Deletion 2 Truncated HIS(SEQ ID NO:68).

Examples of polypetides that can be produced as monomers or rosettes oftrimers include: RSV F furmt Truncated HIS (SEQ ID NO:50); RSV F furdelTruncated HIS (SEQ ID NO: 51); RSV F delP21 furdel Truncated HIS (SEQ IDNO:86); and RSV F delP23 furdel Truncated HIS (SEQ ID NO:49), and RSV FFactor Xa Truncated HIS (SEQ ID NO:52).

An example of a wild-type cleavage that likely produces a rosetteformation is RSV F C-term Furin Truncated HIS (SEQ ID NO:87).

Full

The following polypeptide is a full-length RSV F polypeptide.

(SEQ ID NO: 21)   1MELLILKANA ITTILTAVTF CFASGQNITE EFYQSTCSAV SKGYLSALRT GWYTSVITIE  61LSNIKENKCN GTDAKVKLIK QELDKYKNAV TELQLLMQST PATNNRARRE LPRFMNYTLN 121NAKKTNVTLS KKRKRRFLGF LLGVGSAIAS GVAVSKVLHL EGEVNKIKSA LLSTNKAVVS 181LSNGVSVLTS KVLDLKNYID KQLLPIVNKQ SCSISNIETV IEFQQKNNRL LEITREFSVN 241AGVTTPVSTY MLTNSELLSL INDMPITNDQ KKLMSNNVQI VRQQSYSIMS IIKEEVLAYV 301VQLPLYGVID TPCWKLHTSP LCTTNTKEGS NICLTRTDRG WYCDNAGSVS FFPQAETCKV 361QSNRVFCDTM NSLTLPSEVN LCNVDIFNPK YDCKIMTSKT DVSSSVITSL GAIVSCYGKT 421KCTASNKNRG IIKTFSNGCD YVSNKGVDTV SVGNTLYYVN KQEGKSLYVK GEPIINFYDP 481LVFPSDEFDA SISQVNEKIN QSLAFIRKSD ELLHNVNAGK STTNIMITTI IIVIIVILLS 541LIAVGLLLYC KARSTPVTLS KDQLSGINNI AFSNThe following nucleic acid sequence is the optimized coding sequence forthe foregoing polypeptide sequence.

(SEQ ID NO: 22) 1ATGGAACTGC TGATCCTGAA GGCCAACGCC ATCACCACCA TCCTGACCGC CGTGACCTTC 61TGCTTCGCCA GCGGCCAGAA CATCACCGAG GAATTCTACC AGAGCACCTG CAGCGCCGTG 121AGCAAGGGCT ACCTGAGCGC CCTGCGGACC GGCTGGTACA CCAGCGTGAT CACCATCGAG 181CTGTCCAACA TCAAAGAAAA CAAGTGCAAC GGCACCGACG CCAAGGTGAA ACTGATCAAG 241CAGGAACTGG ACAAGTACAA GAACGCCGTG ACCGAGCTGC AGCTGCTGAT GCAGAGCACC 301CCCGCCACCA ACAACCGGGC CAGAAGAGAG CTGCCCCGGT TCATGAACTA CACCCTGAAC 361AACGCCAAGA AAACCAACGT GACCCTGAGC AAGAAGCGGA AGCGGCGGTT CCTGGGCTTC 421CTGCTGGGCG TGGGCAGCGC CATCGCCAGC GGGGTGGCCG TGTCCAAGGT GCTGCACCTG 481GAAGGCGAGG TGAACAAGAT CAAGTCCGCC CTGCTGTCCA CCAACAAGGC CGTGGTGTCC 541CTGAGCAACG GCGTGAGCGT GCTGACCAGC AAGGTGCTGG ATCTGAAGAA CTACATCGAC 601AAGCAGCTGC TGCCCATCGT GAACAAGCAG AGCTGCAGCA TCAGCAACAT CGAGACCGTG 661ATCGAGTTCC AGCAGAAGAA CAACCGGCTG CTGGAAATCA CCCGGGAGTT CAGCGTGAAC 721GCCGGCGTGA CCACCCCCGT GAGCACCTAC ATGCTGACCA ACAGCGAGCT GCTGTCCCTG 781ATCAATGACA TGCCCATCAC CAACGACCAG AAAAAGCTGA TGAGCAACAA CGTGCAGATC 841GTGCGGCAGC AGAGCTACTC CATCATGAGC ATCATCAAAG AAGAGGTGCT GGCCTACGTG 901GTGCAGCTGC CCCTGTACGG CGTGATCGAC ACCCCCTGCT GGAAGCTGCA CACCAGCCCC 961CTGTGCACCA CCAACACCAA AGAGGGCAGC AACATCTGCC TGACCCGGAC CGACCGGGGC 1021TGGTACTGCG ACAACGCCGG CAGCGTGAGC TTCTTCCCCC AAGCCGAGAC CTGCAAGGTG 1081CAGAGCAACC GGGTGTTCTG CGACACCATG AACAGCCTGA CCCTGCCCTC CGAGGTGAAC 1141CTGTGCAACG TGGACATCTT CAACCCCAAG TACGACTGCA AGATCATGAC CTCCAAGACC 1201GACGTGAGCA GCTCCGTGAT CACCTCCCTG GGCGCCATCG TGAGCTGCTA CGGCAAGACC 1261AAGTGCACCG CCAGCAACAA GAACCGGGGC ATCATCAAGA CCTTCAGCAA CGGCTGCGAC 1321TACGTGAGCA ACAAGGGCGT GGACACCGTG AGCGTGGGCA ACACACTGTA CTACGTGAAT 1381AAGCAGGAAG GCAAGAGCCT GTACGTGAAG GGCGAGCCCA TCATCAACTT CTACGACCCC 1441CTGGTGTTCC CCAGCGACGA GTTCGACGCC AGCATCAGCC AGGTCAACGA GAAGATCAAC 1501CAGAGCCTGG CCTTCATCCG GAAGAGCGAC GAGCTGCTGC ACAATGTGAA TGCCGGCAAG 1561AGCACCACCA ATATCATGAT CACCACAATC ATCATCGTGA TCATTGTGAT CCTGCTGTCT 1621CTGATTGCCG TGGGCCTGCT GCTGTACTGC AAGGCCCGCA GCACCCCTGT GACCCTGTCC 1681AAGGACCAGC TGTCCGGCAT CAACAATATC GCCTTCTCCA ACTGAAG

Full HIS

The following polypeptide includes the full-length RSV F polypeptidefollowed by a hexa-histidine tag.

(SEQ ID NO: 23)   1MELLILKANA ITTILTAVTF CFASGQNITE EFYQSTCSAV SKGYLSALRT GWYTSVITIE  61LSNIKENKCN GTDAKVKLIK QELDKYKNAV TELQLLMQST PATNNRARRE LPRFMNYTLN 121NAKKTNVTLS KKRKRRFLGF LLGVGSAIAS GVAVSKVLHL EGEVNKIKSA LLSTNKAVVS 181LSNGVSVLTS KVLDLKNYID KQLLPIVNKQ SCSISNIETV IEFQQKNNRL LEITREFSVN 241AGVTTPVSTY MLTNSELLSL INDMPITNDQ KKLMSNNVQI VRQQSYSIMS IIKEEVLAYV 301VQLPLYGVID TPCWKLHTSP LCTTNTKEGS NICLTRTDRG WYCDNAGSVS FFPQAETCKV 361QSNRVFCDTM NSLTLPSEVN LCNVDIFNPK YDCKIMTSKT DVSSSVITSL GAIVSCYGKT 421KCTASNKNRG IIKTFSNGCD YVSNKGVDTV SVGNTLYYVN KQEGKSLYVK GEPIINFYDP 481LVFPSDEFDA SISQVNEKIN QSLAFIRKSD ELLHNVNAGK STTNIMITTI IIVIIVILLS 541LIAVGLLLYC KARSTPVTLS KDQLSGINNI AFSNSGGSAG SGHHHHHHThe following nucleic acid sequence is the optimized coding sequence forthe foregoing polypeptide sequence.

(SEQ ID NO: 24) 1ATGGAACTGC TGATCCTGAA GGCCAACGCC ATCACCACCA TCCTGACCGC CGTGACCTTC 61TGCTTCGCCA GCGGCCAGAA CATCACCGAG GAATTCTACC AGAGCACCTG CAGCGCCGTG 121AGCAAGGGCT ACCTGAGCGC CCTGCGGACC GGCTGGTACA CCAGCGTGAT CACCATCGAG 181CTGTCCAACA TCAAAGAAAA CAAGTGCAAC GGCACCGACG CCAAGGTGAA ACTGATCAAG 241CAGGAACTGG ACAAGTACAA GAACGCCGTG ACCGAGCTGC AGCTGCTGAT GCAGAGCACC 301CCCGCCACCA ACAACCGGGC CAGAAGAGAG CTGCCCCGGT TCATGAACTA CACCCTGAAC 361AACGCCAAGA AAACCAACGT GACCCTGAGC AAGAAGCGGA AGCGGCGGTT CCTGGGCTTC 421CTGCTGGGCG TGGGCAGCGC CATCGCCAGC GGGGTGGCCG TGTCCAAGGT GCTGCACCTG 481GAAGGCGAGG TGAACAAGAT CAAGTCCGCC CTGCTGTCCA CCAACAAGGC CGTGGTGTCC 541CTGAGCAACG GCGTGAGCGT GCTGACCAGC AAGGTGCTGG ATCTGAAGAA CTACATCGAC 601AAGCAGCTGC TGCCCATCGT GAACAAGCAG AGCTGCAGCA TCAGCAACAT CGAGACCGTG 661ATCGAGTTCC AGCAGAAGAA CAACCGGCTG CTGGAAATCA CCCGGGAGTT CAGCGTGAAC 721GCCGGCGTGA CCACCCCCGT GAGCACCTAC ATGCTGACCA ACAGCGAGCT GCTGTCCCTG 781ATCAATGACA TGCCCATCAC CAACGACCAG AAAAAGCTGA TGAGCAACAA CGTGCAGATC 841GTGCGGCAGC AGAGCTACTC CATCATGAGC ATCATCAAAG AAGAGGTGCT GGCCTACGTG 901GTGCAGCTGC CCCTGTACGG CGTGATCGAC ACCCCCTGCT GGAAGCTGCA CACCAGCCCC 961CTGTGCACCA CCAACACCAA AGAGGGCAGC AACATCTGCC TGACCCGGAC CGACCGGGGC 1021TGGTACTGCG ACAACGCCGG CAGCGTGAGC TTCTTCCCCC AAGCCGAGAC CTGCAAGGTG 1081CAGAGCAACC GGGTGTTCTG CGACACCATG AACAGCCTGA CCCTGCCCTC CGAGGTGAAC 1141CTGTGCAACG TGGACATCTT CAACCCCAAG TACGACTGCA AGATCATGAC CTCCAAGACC 1201GACGTGAGCA GCTCCGTGAT CACCTCCCTG GGCGCCATCG TGAGCTGCTA CGGCAAGACC 1261AAGTGCACCG CCAGCAACAA GAACCGGGGC ATCATCAAGA CCTTCAGCAA CGGCTGCGAC 1321TACGTGAGCA ACAAGGGCGT GGACACCGTG AGCGTGGGCA ACACACTGTA CTACGTGAAT 1381AAGCAGGAAG GCAAGAGCCT GTACGTGAAG GGCGAGCCCA TCATCAACTT CTACGACCCC 1441CTGGTGTTCC CCAGCGACGA GTTCGACGCC AGCATCAGCC AGGTCAACGA GAAGATCAAC 1501CAGAGCCTGG CCTTCATCCG GAAGAGCGAC GAGCTGCTGC ACAATGTGAA TGCCGGCAAG 1561AGCACCACCA ATATCATGAT CACCACAATC ATCATCGTGA TCATTGTGAT CCTGCTGTCT 1621CTGATTGCCG TGGGCCTGCT GCTGTACTGC AAGGCCCGCA GCACCCCTGT GACCCTGTCC 1681AAGGACCAGC TGTCCGGCAT CAACAATATC GCCTTCTCCA ACAGCGGCGG CAGCGCCGGC 1741TCTGGCCACC ACCACCATCA CCACTGAAG

Full Pre HIS

The following polypeptide includes the full-length RSV F polypeptidewith the trimerization domain of GCN4 (underlined) attached at theC-terminus of the RSV F polypeptide followed by a hexa-histidine tag.

(SEQ ID NO: 25)   1MELLILKANA ITTILTAVTF CFASGQNITE EFYQSTCSAV SKGYLSALRT GWYTSVITIE  61LSNIKENKCN GTDAKVKLIK QELDKYKNAV TELQLLMQST PATNNRARRE LPRFMNYTLN 121NAKKTNVTLS KKRKRRFLGF LLGVGSAIAS GVAVSKVLHL EGEVNKIKSA LLSTNKAVVS 181LSNGVSVLTS KVLDLKNYID KQLLPIVNKQ SCSISNIETV IEFQQKNNRL LEITREFSVN 241AGVTTPVSTY MLTNSELLSL INDMPITNDQ KKLMSNNVQI VRQQSYSIMS IIKEEVLAYV 301VQLPLYGVID TPCWKLHTSP LCTTNTKEGS NICLTRTDRG WYCDNAGSVS FFPQAETCKV 361QSNRVFCDTM NSLTLPSEVN LCNVDIFNPK YDCKIMTSKT DVSSSVITSL GAIVSCYGKT 421KCTASNKNRG IIKTFSNGCD YVSNKGVDTV SVGNTLYYVN KQEGKSLYVK GEPIINFYDP 481LVFPSDEFDA SISQVNEKIN QSLAFIRKSD ELLHNVNAGK STTNIMITTI IIVIIVILLS 541LIAVGLLLYC KARSTPVTLS KDQLSGINNI AFSNGSSGRM KQIEDKIEEI LSKIYHIENE 601IARIKKLIGE SGGSAGSGHH HHHHThe following nucleic acid sequence is the optimized coding sequence forthe foregoing polypeptide sequence.

(SEQ ID NO: 26)    1 ATGGAACTGC TGATCCTGAA GGCCAACGCC     ATCACCACCA TCCTGACCGC CGTGACCTTC  61 TGCTTCGCCA GCGGCCAGAA CATCACCGAG     GAATTCTACC AGAGCACCTG CAGCGCCGTG 121 AGCAAGGGCT ACCTGAGCGC CCTGCGGACC     GGCTGGTACA CCAGCGTGAT CACCATCGAG 181 CTGTCCAACA TCAAAGAAAA CAAGTGCAAC     GGCACCGACG CCAAGGTGAA ACTGATCAAG 241 CAGGAACTGG ACAAGTACAA GAACGCCGTG     ACCGAGCTGC AGCTGCTGAT GCAGAGCACC 301 CCCGCCACCA ACAACCGGGC CAGAAGAGAG     CTGCCCCGGT TCATGAACTA CACCCTGAAC 361 AACGCCAAGA AAACCAACGT GACCCTGAGC     AAGAAGCGGA AGCGGCGGTT CCTGGGCTTC 421 CTGCTGGGCG TGGGCAGCGC CATCGCCAGC     GGGGTGGCCG TGTCCAAGGT GCTGCACCTG 481 GAAGGCGAGG TGAACAAGAT CAAGTCCGCC     CTGCTGTCCA CCAACAAGGC CGTGGTGTCC 541 CTGAGCAACG GCGTGAGCGT GCTGACCAGC     AAGGTGCTGG ATCTGAAGAA CTACATCGAC 601 AAGCAGCTGC TGCCCATCGT GAACAAGCAG     AGCTGCAGCA TCAGCAACAT CGAGACCGTG 661 ATCGAGTTCC AGCAGAAGAA CAACCGGCTG     CTGGAAATCA CCCGGGAGTT CAGCGTGAAC 721 GCCGGCGTGA CCACCCCCGT GAGCACCTAC     ATGCTGACCA ACAGCGAGCT GCTGTCCCTG 781 ATCAATGACA TGCCCATCAC CAACGACCAG     AAAAAGCTGA TGAGCAACAA CGTGCAGATC 841 GTGCGGCAGC AGAGCTACTC CATCATGAGC     ATCATCAAAG AAGAGGTGCT GGCCTACGTG 901 GTGCAGCTGC CCCTGTACGG CGTGATCGAC     ACCCCCTGCT GGAAGCTGCA CACCAGCCCC 961 CTGTGCACCA CCAACACCAA AGAGGGCAGC     AACATCTGCC TGACCCGGAC CGACCGGGGC1021 TGGTACTGCG ACAACGCCGG CAGCGTGAGC     TTCTTCCCCC AAGCCGAGAC CTGCAAGGTG1081 CAGAGCAACC GGGTGTTCTG CGACACCATG     AACAGCCTGA CCCTGCCCTC CGAGGTGAAC1141 CTGTGCAACG TGGACATCTT CAACCCCAAG     TACGACTGCA AGATCATGAC CTCCAAGACC1201 GACGTGAGCA GCTCCGTGAT CACCTCCCTG     GGCGCCATCG TGAGCTGCTA CGGCAAGACC1261 AAGTGCACCG CCAGCAACAA GAACCGGGGC     ATCATCAAGA CCTTCAGCAA CGGCTGCGAC1321 TACGTGAGCA ACAAGGGCGT GGACACCGTG     AGCGTGGGCA ACACACTGTA CTACGTGAAT1381 AAGCAGGAAG GCAAGAGCCT GTACGTGAAG     GGCGAGCCCA TCATCAACTT CTACGACCCC1441 CTGGTGTTCC CCAGCGACGA GTTCGACGCC     AGCATCAGCC AGGTCAACGA GAAGATCAAC1501 CAGAGCCTGG CCTTCATCCG GAAGAGCGAC     GAGCTGCTGC ACAATGTGAA TGCCGGCAAG1561 AGCACCACCA ATATCATGAT CACCACAATC     ATCATCGTGA TCATTGTGAT CCTGCTGTCT1621 CTGATTGCCG TGGGCCTGCT GCTGTACTGC     AAGGCCCGCA GCACCCCTGT GACCCTGTCC1681 AAGGACCAGC TGTCCGGCAT CAACAATATC     GCCTTCTCCA ACGGCAGCAG CGGCCGGATG1741 AAGCAGATCG AGGACAAGAT CGAGGAAATC     CTGAGCAAGA TCTACCACAT CGAGAACGAG1801 ATCGCCCGGA TCAAGAAGCT GATCGGCGAA     AGCGGCGGCT CTGCCGGAAG CGGCCACCAC 1861 CACCATCACC ACTGAAG

Full Pre HIS 2

The following polypeptide includes the full-length RSV F polypeptidewith the trimerization domain of GCN4 (underlined) attached at theC-terminus of the RSV F polypeptide followed by a hexa-histidine tag.

(SEQ ID NO: 27)   1 MELLILKANA ITTILTAVTF CFASGQNITE    EFYQSTCSAV SKGYLSALRT GWYTSVITIE 61 LSNIKENKCN GTDAKVKLIK QELDKYKNAV    TELQLLMQST PATNNRARRE LPRFMNYTLN121 NAKKTNVTLS KKRKRRFLGF LLGVGSAIAS    GVAVSKVLHL EGEVNKIKSA LLSTNKAVVS181 LSNGVSVLTS KVLDLKNYID KQLLPIVNKQ    SCSISNIETV IEFQQKNNRL LEITREFSVN241 AGVTTPVSTY MLTNSELLSL INDMPITNDQ    KKLMSNNVQI VRQQSYSIMS IIKEEVLAYV301 VQLPLYGVID TPCWKLHTSP LCTTNTKEGS    NICLTRTDRG WYCDNAGSVS FFPQAETCKV361 QSNRVFCDTM NSLTLPSEVN LCNVDIFNPK    YDCKIMTSKT DVSSSVITSL GAIVSCYGKT421 KCTASNKNRG IIKTFSNGCD YVSNKGVDTV    SVGNTLYYVN KQEGKSLYVK GEPIINFYDP481 LVFPSDEFDA SISQVNEKIN QSLAFIRKSD    ELLHNVNAGK STTNIMITTI IIVIIVILLS541 LIAVGLLLYC KARSTPVTLS KDQLSGINNI    AFSNGSSGSG RMKQIEDKIE EILSKIYHIE 601 NEIARIKKLI GESGGSAGSG HHHHHHThe following nucleic acid sequence is the optimized coding sequence forthe foregoing polypeptide sequence.

(SEQ ID NO: 28)    1 ATGGAACTGC TGATCCTGAA GGCCAACGCC     ATCACCACCA TCCTGACCGC CGTGACCTTC  61 TGCTTCGCCA GCGGCCAGAA CATCACCGAG     GAATTCTACC AGAGCACCTG CAGCGCCGTG 121 AGCAAGGGCT ACCTGAGCGC CCTGCGGACC     GGCTGGTACA CCAGCGTGAT CACCATCGAG 181 CTGTCCAACA TCAAAGAAAA CAAGTGCAAC     GGCACCGACG CCAAGGTGAA ACTGATCAAG 241 CAGGAACTGG ACAAGTACAA GAACGCCGTG     ACCGAGCTGC AGCTGCTGAT GCAGAGCACC 301 CCCGCCACCA ACAACCGGGC CAGAAGAGAG     CTGCCCCGGT TCATGAACTA CACCCTGAAC 361 AACGCCAAGA AAACCAACGT GACCCTGAGC     AAGAAGCGGA AGCGGCGGTT CCTGGGCTTC 421 CTGCTGGGCG TGGGCAGCGC CATCGCCAGC     GGGGTGGCCG TGTCCAAGGT GCTGCACCTG 481 GAAGGCGAGG TGAACAAGAT CAAGTCCGCC     CTGCTGTCCA CCAACAAGGC CGTGGTGTCC 541 CTGAGCAACG GCGTGAGCGT GCTGACCAGC     AAGGTGCTGG ATCTGAAGAA CTACATCGAC 601 AAGCAGCTGC TGCCCATCGT GAACAAGCAG     AGCTGCAGCA TCAGCAACAT CGAGACCGTG 661 ATCGAGTTCC AGCAGAAGAA CAACCGGCTG     CTGGAAATCA CCCGGGAGTT CAGCGTGAAC 721 GCCGGCGTGA CCACCCCCGT GAGCACCTAC     ATGCTGACCA ACAGCGAGCT GCTGTCCCTG 781 ATCAATGACA TGCCCATCAC CAACGACCAG     AAAAAGCTGA TGAGCAACAA CGTGCAGATC 841 GTGCGGCAGC AGAGCTACTC CATCATGAGC     ATCATCAAAG AAGAGGTGCT GGCCTACGTG 901 GTGCAGCTGC CCCTGTACGG CGTGATCGAC     ACCCCCTGCT GGAAGCTGCA CACCAGCCCC 961 CTGTGCACCA CCAACACCAA AGAGGGCAGC     AACATCTGCC TGACCCGGAC CGACCGGGGC1021 TGGTACTGCG ACAACGCCGG CAGCGTGAGC     TTCTTCCCCC AAGCCGAGAC CTGCAAGGTG1081 CAGAGCAACC GGGTGTTCTG CGACACCATG     AACAGCCTGA CCCTGCCCTC CGAGGTGAAC1141 CTGTGCAACG TGGACATCTT CAACCCCAAG     TACGACTGCA AGATCATGAC CTCCAAGACC1201 GACGTGAGCA GCTCCGTGAT CACCTCCCTG     GGCGCCATCG TGAGCTGCTA CGGCAAGACC1261 AAGTGCACCG CCAGCAACAA GAACCGGGGC     ATCATCAAGA CCTTCAGCAA CGGCTGCGAC1321 TACGTGAGCA ACAAGGGCGT GGACACCGTG     AGCGTGGGCA ACACACTGTA CTACGTGAAT1381 AAGCAGGAAG GCAAGAGCCT GTACGTGAAG     GGCGAGCCCA TCATCAACTT CTACGACCCC1441 CTGGTGTTCC CCAGCGACGA GTTCGACGCC     AGCATCAGCC AGGTCAACGA GAAGATCAAC1501 CAGAGCCTGG CCTTCATCCG GAAGAGCGAC     GAGCTGCTGC ACAATGTGAA TGCCGGCAAG1561 AGCACCACCA ATATCATGAT CACCACAATC     ATCATCGTGA TCATTGTGAT CCTGCTGTCT1621 CTGATTGCCG TGGGCCTGCT GCTGTACTGC     AAGGCCCGCA GCACCCCTGT GACCCTGTCC1681 AAGGACCAGC TGTCCGGCAT CAACAATATC     GCCTTCTCCA ACGGCAGCAG CGGCAGCGGC1741 CGGATGAAGC AGATCGAGGA CAAGATCGAG     GAAATCCTGA GCAAGATCTA CCACATCGAG1801 AACGAGATCG CCCGGATCAA GAAGCTGATC     GGCGAAAGCG GCGGCTCTGC CGGAAGCGGC 1861 CACCACCACC ATCACCACTG AAG

Ecto HIS

The following polypeptide includes the ecto domain of the RSV Fpolypeptide followed by a hexa-histidine tag.

(SEQ ID NO: 29)   1 MELLILKANA ITTILTAVTF CFASGQNITE    EFYQSTCSAV SKGYLSALRT GWYTSVITIE 61 LSNIKENKCN GTDAKVKLIK QELDKYKNAV    TELQLLMQST PATNNRARRE LPRFMNYTLN121 NAKKTNVTLS KKRKRRFLGF LLGVGSAIAS    GVAVSKVLHL EGEVNKIKSA LLSTNKAVVS181 LSNGVSVLTS KVLDLKNYID KQLLPIVNKQ    SCSISNIETV IEFQQKNNRL LEITREFSVN241 AGVTTPVSTY MLTNSELLSL INDMPITNDQ    KKLMSNNVQI VRQQSYSIMS IIKEEVLAYV301 VQLPLYGVID TPCWKLHTSP LCTTNTKEGS    NICLTRTDRG WYCDNAGSVS FFPQAETCKV361 QSNRVFCDTM NSLTLPSEVN LCNVDIFNPK    YDCKIMTSKT DVSSSVITSL GAIVSCYGKT421 KCTASNKNRG IIKTFSNGCD YVSNKGVDTV    SVGNTLYYVN KQEGKSLYVK GEPIINFYDP481 LVFPSDEFDA SISQVNEKIN QSLAFIRKSD     ELLHNVNSGG SAGSGHHHHH HThe following nucleic acid sequence is the optimized coding sequence forthe foregoing polypeptide sequence.

(SEQ ID NO: 30)    1 ATGGAACTGC TGATCCTGAA GGCCAACGCC     ATCACCACCA TCCTGACCGC CGTGACCTTC  61 TGCTTCGCCA GCGGCCAGAA CATCACCGAG     GAATTCTACC AGAGCACCTG CAGCGCCGTG 121 AGCAAGGGCT ACCTGAGCGC CCTGCGGACC     GGCTGGTACA CCAGCGTGAT CACCATCGAG 181 CTGTCCAACA TCAAAGAAAA CAAGTGCAAC     GGCACCGACG CCAAGGTGAA ACTGATCAAG 241 CAGGAACTGG ACAAGTACAA GAACGCCGTG     ACCGAGCTGC AGCTGCTGAT GCAGAGCACC 301 CCCGCCACCA ACAACCGGGC CAGAAGAGAG     CTGCCCCGGT TCATGAACTA CACCCTGAAC 361 AACGCCAAGA AAACCAACGT GACCCTGAGC     AAGAAGCGGA AGCGGCGGTT CCTGGGCTTC 421 CTGCTGGGCG TGGGCAGCGC CATCGCCAGC     GGGGTGGCCG TGTCCAAGGT GCTGCACCTG 481 GAAGGCGAGG TGAACAAGAT CAAGTCCGCC     CTGCTGTCCA CCAACAAGGC CGTGGTGTCC 541 CTGAGCAACG GCGTGAGCGT GCTGACCAGC     AAGGTGCTGG ATCTGAAGAA CTACATCGAC 601 AAGCAGCTGC TGCCCATCGT GAACAAGCAG     AGCTGCAGCA TCAGCAACAT CGAGACCGTG 661 ATCGAGTTCC AGCAGAAGAA CAACCGGCTG     CTGGAAATCA CCCGGGAGTT CAGCGTGAAC 721 GCCGGCGTGA CCACCCCCGT GAGCACCTAC     ATGCTGACCA ACAGCGAGCT GCTGTCCCTG 781 ATCAATGACA TGCCCATCAC CAACGACCAG     AAAAAGCTGA TGAGCAACAA CGTGCAGATC 841 GTGCGGCAGC AGAGCTACTC CATCATGAGC     ATCATCAAAG AAGAGGTGCT GGCCTACGTG 901 GTGCAGCTGC CCCTGTACGG CGTGATCGAC     ACCCCCTGCT GGAAGCTGCA CACCAGCCCC 961 CTGTGCACCA CCAACACCAA AGAGGGCAGC     AACATCTGCC TGACCCGGAC CGACCGGGGC1021 TGGTACTGCG ACAACGCCGG CAGCGTGAGC     TTCTTCCCCC AAGCCGAGAC CTGCAAGGTG1081 CAGAGCAACC GGGTGTTCTG CGACACCATG     AACAGCCTGA CCCTGCCCTC CGAGGTGAAC1141 CTGTGCAACG TGGACATCTT CAACCCCAAG     TACGACTGCA AGATCATGAC CTCCAAGACC1201 GACGTGAGCA GCTCCGTGAT CACCTCCCTG     GGCGCCATCG TGAGCTGCTA CGGCAAGACC1261 AAGTGCACCG CCAGCAACAA GAACCGGGGC     ATCATCAAGA CCTTCAGCAA CGGCTGCGAC1321 TACGTGAGCA ACAAGGGCGT GGACACCGTG     AGCGTGGGCA ACACACTGTA CTACGTGAAT1381 AAGCAGGAAG GCAAGAGCCT GTACGTGAAG     GGCGAGCCCA TCATCAACTT CTACGACCCC1441 CTGGTGTTCC CCAGCGACGA GTTCGACGCC     AGCATCAGCC AGGTCAACGA GAAGATCAAC1501 CAGAGCCTGG CCTTCATCCG GAAGAGCGAC     GAGCTGCTGC ACAATGTGAA TAGCGGCGGC1561 AGCGCCGGCT CTGGCCACCA CCACCATCAC      CACTGAAG

Ecto Pre HIS

The following polypeptide includes the ecto domain of the RSV Fpolypeptide with the trimerization domain of GCN4 (underlined) insertedinto the RSV F polypeptide up stream of where the TM domain of the RSVprotein would have been (beginning at a.a. 517) followed by ahexa-histidine tag.

(SEQ ID NO: 31)   1 MELLILKANA ITTILTAVTF CFASGQNITE    EFYQSTCSAV SKGYLSALRT GWYTSVITIE 61 LSNIKENKCN GTDAKVKLIK QELDKYKNAV    TELQLLMQST PATNNRARRE LPRFMNYTLN121 NAKKTNVTLS KKRKRRFLGF LLGVGSAIAS     GVAVSKVLHL EGEVNKIKSA LLSTNKAWS181 LSNGVSVLTS KVLDLKNYID KQLLPIVNKQ    SCSISNIETV IEFQQKNNRL LEITREFSVN241 AGVTTPVSTY MLTNSELLSL INDMPITNDQ    KKLMSNNVQI VRQQSYSIMS IIKEEVLAYV301 VQLPLYGVID TPCWKLHTSP LCTTNTKEGS    NICLTRTDRG WYCDNAGSVS FFPQAETCKV361 QSNRVFCDTM NSLTLPSEVN LCNVDIFNPK    YDCKIMTSKT DVSSSVITSL GAIVSCYGKT421 KCTASNKNRG IIKTFSNGCD YVSNKGVDTV    SVGNTLYYVN KQEGKSLYVK GEPIINFYDP481 LVFPSDEFDA SISQVNEKIN QSLAFIRKSD    ELLHNVNDKI EEILSKIYHI ENEIARIKKL 541 IGESGGSAGS GHHHHHHThe following nucleic acid sequence is the optimized coding sequence forthe foregoing polypeptide sequence.

(SEQ ID NO: 32)    1 ATGGAACTGC TGATCCTGAA GGCCAACGCC     ATCACCACCA TCCTGACCGC CGTGACCTTC  61 TGCTTCGCCA GCGGCCAGAA CATCACCGAG     GAATTCTACC AGAGCACCTG CAGCGCCGTG 121 AGCAAGGGCT ACCTGAGCGC CCTGCGGACC     GGCTGGTACA CCAGCGTGAT CACCATCGAG 181 CTGTCCAACA TCAAAGAAAA CAAGTGCAAC     GGCACCGACG CCAAGGTGAA ACTGATCAAG 241 CAGGAACTGG ACAAGTACAA GAACGCCGTG     ACCGAGCTGC AGCTGCTGAT GCAGAGCACC 301 CCCGCCACCA ACAACCGGGC CAGAAGAGAG     CTGCCCCGGT TCATGAACTA CACCCTGAAC 361 AACGCCAAGA AAACCAACGT GACCCTGAGC     AAGAAGCGGA AGCGGCGGTT CCTGGGCTTC 421 CTGCTGGGCG TGGGCAGCGC CATCGCCAGC     GGGGTGGCCG TGTCCAAGGT GCTGCACCTG 481 GAAGGCGAGG TGAACAAGAT CAAGTCCGCC     CTGCTGTCCA CCAACAAGGC CGTGGTGTCC 541 CTGAGCAACG GCGTGAGCGT GCTGACCAGC     AAGGTGCTGG ATCTGAAGAA CTACATCGAC 601 AAGCAGCTGC TGCCCATCGT GAACAAGCAG     AGCTGCAGCA TCAGCAACAT CGAGACCGTG 661 ATCGAGTTCC AGCAGAAGAA CAACCGGCTG     CTGGAAATCA CCCGGGAGTT CAGCGTGAAC 721 GCCGGCGTGA CCACCCCCGT GAGCACCTAC     ATGCTGACCA ACAGCGAGCT GCTGTCCCTG 781 ATCAATGACA TGCCCATCAC CAACGACCAG     AAAAAGCTGA TGAGCAACAA CGTGCAGATC 841 GTGCGGCAGC AGAGCTACTC CATCATGAGC     ATCATCAAAG AAGAGGTGCT GGCCTACGTG 901 GTGCAGCTGC CCCTGTACGG CGTGATCGAC     ACCCCCTGCT GGAAGCTGCA CACCAGCCCC 961 CTGTGCACCA CCAACACCAA AGAGGGCAGC     AACATCTGCC TGACCCGGAC CGACCGGGGC1021 TGGTACTGCG ACAACGCCGG CAGCGTGAGC     TTCTTCCCCC AAGCCGAGAC CTGCAAGGTG1081 CAGAGCAACC GGGTGTTCTG CGACACCATG     AACAGCCTGA CCCTGCCCTC CGAGGTGAAC1141 CTGTGCAACG TGGACATCTT CAACCCCAAG     TACGACTGCA AGATCATGAC CTCCAAGACC1201 GACGTGAGCA GCTCCGTGAT CACCTCCCTG     GGCGCCATCG TGAGCTGCTA CGGCAAGACC1261 AAGTGCACCG CCAGCAACAA GAACCGGGGC     ATCATCAAGA CCTTCAGCAA CGGCTGCGAC1321 TACGTGAGCA ACAAGGGCGT GGACACCGTG     AGCGTGGGCA ACACACTGTA CTACGTGAAT1381 AAGCAGGAAG GCAAGAGCCT GTACGTGAAG     GGCGAGCCCA TCATCAACTT CTACGACCCC1441 CTGGTGTTCC CCAGCGACGA GTTCGACGCC     AGCATCAGCC AGGTCAACGA GAAGATCAAC1501 CAGAGCCTGG CCTTCATCCG GAAGAGCGAC     GAGCTGCTGC ACAATGTGAA TGACAAGATC1561 GAGGAAATCC TGAGCAAGAT CTACCACATC     GAGAACGAGA TCGCCCGGAT CAAGAAGCTG1621 ATCGGCGAAA GCGGCGGCTC TGCCGGAAGC      GGCCACCACC ACCATCACCA CTGAAG

Full Pre HA HIS

The following polypeptide includes the full-length RSV F polypeptidewith the post-fusion trimerization domain of the influenza hemagglutininpolypeptide (underlined) attached at the C-terminus of the RSV Fpolypeptide followed by a hexa-histidine tag.

(SEQ ID NO: 33)   1 MELLILKANA ITTILTAVTF CFASGQNITE    EFYQSTCSAV SKGYLSALRT GWYTSVITIE 61 LSNIKENKCN GTDAKVKLIK QELDKYKNAV    TELQLLMQST PATNNRARRE LPRFMNYTLN121 NAKKTNVTLS KKRKRRFLGF LLGVGSAIAS     GVAVSKVLHL EGEVNKIKSA LLSTNKAWS181 LSNGVSVLTS KVLDLKNYID KQLLPIVNKQ    SCSISNIETV IEFQQKNNRL LEITREFSVN241 AGVTTPVSTY MLTNSELLSL INDMPITNDQ    KKLMSNNVQI VRQQSYSIMS IIKEEVLAYV301 VQLPLYGVID TPCWKLHTSP LCTTNTKEGS    NICLTRTDRG WYCDNAGSVS FFPQAETCKV361 QSNRVFCDTM NSLTLPSEVN LCNVDIFNPK    YDCKIMTSKT DVSSSVITSL GAIVSCYGKT421 KCTASNKNRG IIKTFSNGCD YVSNKGVDTV    SVGNTLYYVN KQEGKSLYVK GEPIINFYDP481 LVFPSDEFDA SISQVNEKIN QSLAFIRKSD    ELLHNVNAGK STTNIMITTI IIVIIVILLS541 LIAVGLLLYC KARSTPVTLS KDQLSGINNI    AFSNGSSGNE KFHQIEKEFS EVEGRIQDLE 601 KSGGSAGSGH HHHHHThe following nucleic acid sequence is the optimized coding sequence forthe foregoing polypeptide sequence.

(SEQ ID NO: 34)    1 ATGGAACTGC TGATCCTGAA GGCCAACGCC     ATCACCACCA TCCTGACCGC CGTGACCTTC  61 TGCTTCGCCA GCGGCCAGAA CATCACCGAG     GAATTCTACC AGAGCACCTG CAGCGCCGTG 121 AGCAAGGGCT ACCTGAGCGC CCTGCGGACC     GGCTGGTACA CCAGCGTGAT CACCATCGAG 181 CTGTCCAACA TCAAAGAAAA CAAGTGCAAC     GGCACCGACG CCAAGGTGAA ACTGATCAAG 241 CAGGAACTGG ACAAGTACAA GAACGCCGTG     ACCGAGCTGC AGCTGCTGAT GCAGAGCACC 301 CCCGCCACCA ACAACCGGGC CAGAAGAGAG     CTGCCCCGGT TCATGAACTA CACCCTGAAC 361 AACGCCAAGA AAACCAACGT GACCCTGAGC     AAGAAGCGGA AGCGGCGGTT CCTGGGCTTC 421 CTGCTGGGCG TGGGCAGCGC CATCGCCAGC     GGGGTGGCCG TGTCCAAGGT GCTGCACCTG 481 GAAGGCGAGG TGAACAAGAT CAAGTCCGCC     CTGCTGTCCA CCAACAAGGC CGTGGTGTCC 541 CTGAGCAACG GCGTGAGCGT GCTGACCAGC     AAGGTGCTGG ATCTGAAGAA CTACATCGAC 601 AAGCAGCTGC TGCCCATCGT GAACAAGCAG     AGCTGCAGCA TCAGCAACAT CGAGACCGTG 661 ATCGAGTTCC AGCAGAAGAA CAACCGGCTG     CTGGAAATCA CCCGGGAGTT CAGCGTGAAC 721 GCCGGCGTGA CCACCCCCGT GAGCACCTAC     ATGCTGACCA ACAGCGAGCT GCTGTCCCTG 781 ATCAATGACA TGCCCATCAC CAACGACCAG     AAAAAGCTGA TGAGCAACAA CGTGCAGATC 841 GTGCGGCAGC AGAGCTACTC CATCATGAGC     ATCATCAAAG AAGAGGTGCT GGCCTACGTG 901 GTGCAGCTGC CCCTGTACGG CGTGATCGAC     ACCCCCTGCT GGAAGCTGCA CACCAGCCCC 961 CTGTGCACCA CCAACACCAA AGAGGGCAGC     AACATCTGCC TGACCCGGAC CGACCGGGGC1021 TGGTACTGCG ACAACGCCGG CAGCGTGAGC     TTCTTCCCCC AAGCCGAGAC CTGCAAGGTG1081 CAGAGCAACC GGGTGTTCTG CGACACCATG     AACAGCCTGA CCCTGCCCTC CGAGGTGAAC1141 CTGTGCAACG TGGACATCTT CAACCCCAAG     TACGACTGCA AGATCATGAC CTCCAAGACC1201 GACGTGAGCA GCTCCGTGAT CACCTCCCTG     GGCGCCATCG TGAGCTGCTA CGGCAAGACC1261 AAGTGCACCG CCAGCAACAA GAACCGGGGC     ATCATCAAGA CCTTCAGCAA CGGCTGCGAC1321 TACGTGAGCA ACAAGGGCGT GGACACCGTG     AGCGTGGGCA ACACACTGTA CTACGTGAAT1381 AAGCAGGAAG GCAAGAGCCT GTACGTGAAG     GGCGAGCCCA TCATCAACTT CTACGACCCC1441 CTGGTGTTCC CCAGCGACGA GTTCGACGCC     AGCATCAGCC AGGTCAACGA GAAGATCAAC1501 CAGAGCCTGG CCTTCATCCG GAAGAGCGAC     GAGCTGCTGC ACAATGTGAA TGCCGGCAAG1561 AGCACCACCA ATATCATGAT CACCACAATC     ATCATCGTGA TCATTGTGAT CCTGCTGTCT1621 CTGATTGCCG TGGGCCTGCT GCTGTACTGC     AAGGCCCGCA GCACCCCTGT GACCCTGTCC1681 AAGGACCAGC TGTCCGGCAT CAACAATATC     GCCTTCTCCA ACGGCAGCAG CGGCAATGAG1741 AAGTTCCACC AGATCGAGAA AGAATTCAGC     GAGGTGGAGG GCCGGATCCA GGACCTGGAA1801 AAGAGCGGCG GCTCTGCCGG AAGCGGCCAC      CACCACCATC ACCACTGAAG

Ecto Pre HA HIS

The following polypeptide includes the ecto domain of the RSV Fpolypeptide with the post-fusion trimerization domain of the influenzahemagglutinin polypeptide (underlined) inserted into the RSV Fpolypeptide up stream of where the TM domain of the RSV protein wouldhave been (beginning at a.a. 517) followed by a hexa-histidine tag.

(SEQ ID NO: 35)   1 MELLILKANA ITTILTAVTF CFASGQNITE    EFYQSTCSAV SKGYLSALRT GWYTSVITIE 61 LSNIKENKCN GTDAKVKLIK QELDKYKNAV    TELQLLMQST PATNNRARRE LPRFMNYTLN121 NAKKTNVTLS KKRKRRFLGF LLGVGSAIAS    GVAVSKVLHL EGEVNKIKSA LLSTNKAVVS181 LSNGVSVLTS KVLDLKNYID KQLLPIVNKQ    SCSISNIETV IEFQQKNNRL LEITREFSVN241 AGVTTPVSTY MLTNSELLSL INDMPITNDQ    KKLMSNNVQI VRQQSYSIMS IIKEEVLAYV301 VQLPLYGVID TPCWKLHTSP LCTTNTKEGS    NICLTRTDRG WYCDNAGSVS FFPQAETCKV361 QSNRVFCDTM NSLTLPSEVN LCNVDIFNPK    YDCKIMTSKT DVSSSVITSL GAIVSCYGKT421 KCTASNKNRG IIKTFSNGCD YVSNKGVDTV    SVGNTLYYVN KQEGKSLYVK GEPIINFYDP481 LVFPSDEFDA SISQVNEKIN QSLAFIRKSD    ELLHNVNEKF HQIEKEFSEV EGRIQDLEKS 541 GGSAGSGHHH HHH

The following nucleic acid sequence is the optimized coding sequence forthe foregoing polypeptide sequence.

(SEQ ID NO: 36)    1 ATGGAACTGC TGATCCTGAA GGCCAACGCC     ATCACCACCA TCCTGACCGC CGTGACCTTC  61 TGCTTCGCCA GCGGCCAGAA CATCACCGAG     GAATTCTACC AGAGCACCTG CAGCGCCGTG 121 AGCAAGGGCT ACCTGAGCGC CCTGCGGACC     GGCTGGTACA CCAGCGTGAT CACCATCGAG 181 CTGTCCAACA TCAAAGAAAA CAAGTGCAAC     GGCACCGACG CCAAGGTGAA ACTGATCAAG 241 CAGGAACTGG ACAAGTACAA GAACGCCGTG     ACCGAGCTGC AGCTGCTGAT GCAGAGCACC 301 CCCGCCACCA ACAACCGGGC CAGAAGAGAG     CTGCCCCGGT TCATGAACTA CACCCTGAAC 361 AACGCCAAGA AAACCAACGT GACCCTGAGC     AAGAAGCGGA AGCGGCGGTT CCTGGGCTTC 421 CTGCTGGGCG TGGGCAGCGC CATCGCCAGC     GGGGTGGCCG TGTCCAAGGT GCTGCACCTG 481 GAAGGCGAGG TGAACAAGAT CAAGTCCGCC     CTGCTGTCCA CCAACAAGGC CGTGGTGTCC 541 CTGAGCAACG GCGTGAGCGT GCTGACCAGC     AAGGTGCTGG ATCTGAAGAA CTACATCGAC 601 AAGCAGCTGC TGCCCATCGT GAACAAGCAG     AGCTGCAGCA TCAGCAACAT CGAGACCGTG 661 ATCGAGTTCC AGCAGAAGAA CAACCGGCTG     CTGGAAATCA CCCGGGAGTT CAGCGTGAAC 721 GCCGGCGTGA CCACCCCCGT GAGCACCTAC     ATGCTGACCA ACAGCGAGCT GCTGTCCCTG 781 ATCAATGACA TGCCCATCAC CAACGACCAG     AAAAAGCTGA TGAGCAACAA CGTGCAGATC 841 GTGCGGCAGC AGAGCTACTC CATCATGAGC     ATCATCAAAG AAGAGGTGCT GGCCTACGTG 901 GTGCAGCTGC CCCTGTACGG CGTGATCGAC     ACCCCCTGCT GGAAGCTGCA CACCAGCCCC 961 CTGTGCACCA CCAACACCAA AGAGGGCAGC     AACATCTGCC TGACCCGGAC CGACCGGGGC1021 TGGTACTGCG ACAACGCCGG CAGCGTGAGC     TTCTTCCCCC AAGCCGAGAC CTGCAAGGTG1081 CAGAGCAACC GGGTGTTCTG CGACACCATG     AACAGCCTGA CCCTGCCCTC CGAGGTGAAC1141 CTGTGCAACG TGGACATCTT CAACCCCAAG     TACGACTGCA AGATCATGAC CTCCAAGACC1201 GACGTGAGCA GCTCCGTGAT CACCTCCCTG     GGCGCCATCG TGAGCTGCTA CGGCAAGACC1261 AAGTGCACCG CCAGCAACAA GAACCGGGGC     ATCATCAAGA CCTTCAGCAA CGGCTGCGAC1321 TACGTGAGCA ACAAGGGCGT GGACACCGTG     AGCGTGGGCA ACACACTGTA CTACGTGAAT1381 AAGCAGGAAG GCAAGAGCCT GTACGTGAAG     GGCGAGCCCA TCATCAACTT CTACGACCCC1441 CTGGTGTTCC CCAGCGACGA GTTCGACGCC     AGCATCAGCC AGGTCAACGA GAAGATCAAC1501 CAGAGCCTGG CCTTCATCCG GAAGAGCGAC     GAGCTGCTGC ACAATGTGAA TGAGAAGTTC1561 CACCAGATCG AGAAAGAATT CAGCGAGGTG     GAGGGCCGGA TCCAGGACCT GGAAAAGAGC1621 GGCGGCTCTG CCGGAAGCGG CCACCACCAC      CATCACCACT GAAG

FullΔHRB HIS

The following polypeptide includes the full-length RSV F polypeptidewith the HRB domain deleted followed by a hexa-histidine tag.

(SEQ ID NO: 37)   1 MELLILKANA ITTILTAVTF CFASGQNITE    EFYQSTCSAV SKGYLSALRT GWYTSVITIE 61 LSNIKENKCN GTDAKVKLIK QELDKYKNAV    TELQLLMQST PATNNRARRE LPRFMNYTLN121 NAKKTNVTLS KKRKRRFLGF LLGVGSAIAS    GVAVSKVLHL EGEVNKIKSA LLSTNKAVVS181 LSNGVSVLTS KVLDLKNYID KQLLPIVNKQ    SCSISNIETV IEFQQKNNRL LEITREFSVN241 AGVTTPVSTY MLTNSELLSL INDMPITNDQ    KKLMSNNVQI VRQQSYSIMS IIKEEVLAYV301 VQLPLYGVID TPCWKLHTSP LCTTNTKEGS    NICLTRTDRG WYCDNAGSVS FFPQAETCKV361 QSNRVFCDTM NSLTLPSEVN LCNVDIFNPK    YDCKIMTSKT DVSSSVITSL GAIVSCYGKT421 KCTASNKNRG IIKTFSNGCD YVSNKGVDTV    SVGNTLYYVN KQEGKSLYVK GEPNIMITTI481 IIVIIVILLS LIAVGLLLYC KARSTPVTLS     KDQLSGINNI AFSNMGGSHH HHHH

The following nucleic acid sequence is the optimized coding sequence forthe foregoing polypeptide sequence.

(SEQ ID NO: 38)    1 ATGGAACTGC TGATCCTGAA GGCCAACGCC     ATCACCACCA TCCTGACCGC CGTGACCTTC  61 TGCTTCGCCA GCGGCCAGAA CATCACCGAG     GAATTCTACC AGAGCACCTG CAGCGCCGTG 121 AGCAAGGGCT ACCTGAGCGC CCTGCGGACC     GGCTGGTACA CCAGCGTGAT CACCATCGAG 181 CTGTCCAACA TCAAAGAAAA CAAGTGCAAC     GGCACCGACG CCAAGGTGAA ACTGATCAAG 241 CAGGAACTGG ACAAGTACAA GAACGCCGTG     ACCGAGCTGC AGCTGCTGAT GCAGAGCACC 301 CCCGCCACCA ACAACCGGGC CAGAAGAGAG     CTGCCCCGGT TCATGAACTA CACCCTGAAC 361 AACGCCAAGA AAACCAACGT GACCCTGAGC     AAGAAGCGGA AGCGGCGGTT CCTGGGCTTC 421 CTGCTGGGCG TGGGCAGCGC CATCGCCAGC     GGGGTGGCCG TGTCCAAGGT GCTGCACCTG 481 GAAGGCGAGG TGAACAAGAT CAAGTCCGCC     CTGCTGTCCA CCAACAAGGC CGTGGTGTCC 541 CTGAGCAACG GCGTGAGCGT GCTGACCAGC     AAGGTGCTGG ATCTGAAGAA CTACATCGAC 601 AAGCAGCTGC TGCCCATCGT GAACAAGCAG     AGCTGCAGCA TCAGCAACAT CGAGACCGTG 661 ATCGAGTTCC AGCAGAAGAA CAACCGGCTG     CTGGAAATCA CCCGGGAGTT CAGCGTGAAC 721 GCCGGCGTGA CCACCCCCGT GAGCACCTAC     ATGCTGACCA ACAGCGAGCT GCTGTCCCTG 781 ATCAATGACA TGCCCATCAC CAACGACCAG     AAAAAGCTGA TGAGCAACAA CGTGCAGATC 841 GTGCGGCAGC AGAGCTACTC CATCATGAGC     ATCATCAAAG AAGAGGTGCT GGCCTACGTG 901 GTGCAGCTGC CCCTGTACGG CGTGATCGAC     ACCCCCTGCT GGAAGCTGCA CACCAGCCCC 961 CTGTGCACCA CCAACACCAA AGAGGGCAGC     AACATCTGCC TGACCCGGAC CGACCGGGGC1021 TGGTACTGCG ACAACGCCGG CAGCGTGAGC     TTCTTCCCCC AAGCCGAGAC CTGCAAGGTG1081 CAGAGCAACC GGGTGTTCTG CGACACCATG     AACAGCCTGA CCCTGCCCTC CGAGGTGAAC1141 CTGTGCAACG TGGACATCTT CAACCCCAAG     TACGACTGCA AGATCATGAC CTCCAAGACC1201 GACGTGAGCA GCTCCGTGAT CACCTCCCTG     GGCGCCATCG TGAGCTGCTA CGGCAAGACC1261 AAGTGCACCG CCAGCAACAA GAACCGGGGC     ATCATCAAGA CCTTCAGCAA CGGCTGCGAC1321 TACGTGAGCA ACAAGGGCGT GGACACCGTG     AGCGTGGGCA ACACACTGTA CTACGTGAAT1381 AAGCAGGAAG GCAAGAGCCT GTACGTGAAG     GGCGAGCCCA ATATCATGAT CACCACAATC1441 ATCATCGTGA TCATTGTGAT CCTGCTGTCT     CTGATTGCCG TGGGCCTGCT GCTGTACTGC1501 AAGGCCCGCA GCACCCCTGT GACCCTGTCC     AAGGACCAGC TGTCCGGCAT CAACAATATC1561 GCCTTCTCCA ACATGGGGGG TTCTCATCAT      CATCATCATC ATTGAAG

Ecto

The following polypeptide includes just the ecto domain of the RSV Fpolypeptide.

(SEQ ID NO: 39)   1 MELLILKANA ITTILTAVTF CFASGQNITE    EFYQSTCSAV SKGYLSALRT GWYTSVITIE 61 LSNIKENKCN GTDAKVKLIK QELDKYKNAV    TELQLLMQST PATNNRARRE LPRFMNYTLN121 NAKKTNVTLS KKRKRRFLGF LLGVGSAIAS    GVAVSKVLHL EGEVNKIKSA LLSTNKAVVS181 LSNGVSVLTS KVLDLKNYID KQLLPIVNKQ    SCSISNIETV IEFQQKNNRL LEITREFSVN241 AGVTTPVSTY MLTNSELLSL INDMPITNDQ    KKLMSNNVQI VRQQSYSIMS IIKEEVLAYV301 VQLPLYGVID TPCWKLHTSP LCTTNTKEGS    NICLTRTDRG WYCDNAGSVS FFPQAETCKV361 QSNRVFCDTM NSLTLPSEVN LCNVDIFNPK    YDCKIMTSKT DVSSSVITSL GAIVSCYGKT421 KCTASNKNRG IIKTFSNGCD YVSNKGVDTV    SVGNTLYYVN KQEGKSLYVK GEPIINFYDP481 LVFPSDEFDA SISQVNEKIN QSLAFIRKSD     ELLHNVNAGK STTN

The following nucleic acid sequence is the optimized coding sequence forthe foregoing polypeptide sequence.

(SEQ ID NO: 40)    1 ATGGAACTGC TGATCCTGAA GGCCAACGCC     ATCACCACCA TCCTGACCGC CGTGACCTTC  61 TGCTTCGCCA GCGGCCAGAA CATCACCGAG     GAATTCTACC AGAGCACCTG CAGCGCCGTG 121 AGCAAGGGCT ACCTGAGCGC CCTGCGGACC     GGCTGGTACA CCAGCGTGAT CACCATCGAG 181 CTGTCCAACA TCAAAGAAAA CAAGTGCAAC     GGCACCGACG CCAAGGTGAA ACTGATCAAG 241 CAGGAACTGG ACAAGTACAA GAACGCCGTG     ACCGAGCTGC AGCTGCTGAT GCAGAGCACC 301 CCCGCCACCA ACAACCGGGC CAGAAGAGAG     CTGCCCCGGT TCATGAACTA CACCCTGAAC 361 AACGCCAAGA AAACCAACGT GACCCTGAGC     AAGAAGCGGA AGCGGCGGTT CCTGGGCTTC 421 CTGCTGGGCG TGGGCAGCGC CATCGCCAGC     GGGGTGGCCG TGTCCAAGGT GCTGCACCTG 481 GAAGGCGAGG TGAACAAGAT CAAGTCCGCC     CTGCTGTCCA CCAACAAGGC CGTGGTGTCC 541 CTGAGCAACG GCGTGAGCGT GCTGACCAGC     AAGGTGCTGG ATCTGAAGAA CTACATCGAC 601 AAGCAGCTGC TGCCCATCGT GAACAAGCAG     AGCTGCAGCA TCAGCAACAT CGAGACCGTG 661 ATCGAGTTCC AGCAGAAGAA CAACCGGCTG     CTGGAAATCA CCCGGGAGTT CAGCGTGAAC 721 GCCGGCGTGA CCACCCCCGT GAGCACCTAC     ATGCTGACCA ACAGCGAGCT GCTGTCCCTG 781 ATCAATGACA TGCCCATCAC CAACGACCAG     AAAAAGCTGA TGAGCAACAA CGTGCAGATC 841 GTGCGGCAGC AGAGCTACTC CATCATGAGC     ATCATCAAAG AAGAGGTGCT GGCCTACGTG 901 GTGCAGCTGC CCCTGTACGG CGTGATCGAC     ACCCCCTGCT GGAAGCTGCA CACCAGCCCC 961 CTGTGCACCA CCAACACCAA AGAGGGCAGC     AACATCTGCC TGACCCGGAC CGACCGGGGC1021 TGGTACTGCG ACAACGCCGG CAGCGTGAGC     TTCTTCCCCC AAGCCGAGAC CTGCAAGGTG1081 CAGAGCAACC GGGTGTTCTG CGACACCATG     AACAGCCTGA CCCTGCCCTC CGAGGTGAAC1141 CTGTGCAACG TGGACATCTT CAACCCCAAG     TACGACTGCA AGATCATGAC CTCCAAGACC1201 GACGTGAGCA GCTCCGTGAT CACCTCCCTG     GGCGCCATCG TGAGCTGCTA CGGCAAGACC1261 AAGTGCACCG CCAGCAACAA GAACCGGGGC     ATCATCAAGA CCTTCAGCAA CGGCTGCGAC1321 TACGTGAGCA ACAAGGGCGT GGACACCGTG     AGCGTGGGCA ACACACTGTA CTACGTGAAT1381 AAGCAGGAAG GCAAGAGCCT GTACGTGAAG     GGCGAGCCCA TCATCAACTT CTACGACCCC1441 CTGGTGTTCC CCAGCGACGA GTTCGACGCC     AGCATCAGCC AGGTCAACGA GAAGATCAAC1501 CAGAGCCTGG CCTTCATCCG GAAGAGCGAC     GAGCTGCTGC ACAATGTGAA TGCCGGCAAG 1561 AGCACCACCA ATTGAAGRSV F Full Length (SEQ ID NO: 41) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCN GTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGF LLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQ SCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQI VRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVS FFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKT KCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDA SISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLS KDQLSGINNIAFSNRSV F Cleavage Enterokinase idealized (SEQ ID NO: 42)MELLILKANAITTILTAVTFCFASGQNITEEFYQS TCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNN RARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTN KAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTT PVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWK LHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVD IFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNT LYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQINEKINQILAFIRKIDELLHNINAGKSTTNG SGSGDDDDDKGSGSGIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN RSV F Cleavage Thrombin idealized(SEQ ID NO: 43) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCN GTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGF LLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQ SCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQI VRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVS FFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKT KCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDA SISQINEKINQILAFIRKIDELLHNINAGKSTTNGSGSGLVPRGSGSGIMITTIIIVIIVILLSLIAVGL LLYCKARSTPVTLSKDQLSGINNIAFSNRSV F Cleavage FactorXa idealized (SEQ ID NO: 44)MELLILKANAITTILTAVTFCFASGQNITEEFYQS TCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNN RARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTN KAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTT PVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWK LHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVD IFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNT LYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQINEKINQILAFIRKIDELLHNINAGKSTTNG SGSGIEGRGSGSGIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN RSV F furx Truncated HIS (SEQ ID NO: 45)MELLILKANAITTILTAVTFCFASGQNITEEFYQS TCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNN QAQNELPRFMNYTLNNAKKTNVTLSQNQNQNFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTN KAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTT PVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWK LHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVD IFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNT LYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNG GSAGSGHHHHHHRSV F furx R113Q, K123N, K124N Truncated HIS (SEQ ID NO: 46)MELLILKANAITTILTAVTFCFASGQNITEEFYQS TCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNN QAQNELPQFMNYTLNNANNTNVTLSQNQNQNFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTN KAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTT PVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWK LHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVD IFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNT LYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNG GSAGSGHHHHHHRSV F furx R113Q, K123Q, K124Q Truncated HIS (SEQ ID NO: 93)MELLILKANAITTILTAVTFCFASGQNITEEFYQS TCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNN QAQNELPQFMNYTLNNAQQTNVTLSQNQNQNFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTN KAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTT PVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWK LHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVD IFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNT LYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNG GSAGSGHHHHHHRSV F delP21 furx Truncated HIS (SEQ ID NO: 47)MELLILKANAITTILTAVTFCFASGQNITEEFYQS TCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNN QAQN---------------------QNQNQNFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTN KAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTT PVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWK LHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVD IFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNT LYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNG GSAGSGHHHHHH(the symbol “-” idicates that the amino acidat this position is deleted) RSV F delP23 furx Truncated HIS(SEQ ID NO: 48) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCN GTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNQAQN---------------------QNQNFLGFLL GVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSC SISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVR QQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFF PQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKC TASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASI SQVNEKINQSLAFIRKSDELLHNVNAGKSTTNGGSAGSGHHHHHH (the the symbol “-” idicates that theamino acid at this position is deleted)RSV F delP23 furdel Truncated HIS (SEQ ID NO: 49)MELLILKANAITTILTAVTFCFASGQNITEEFYQS TCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNN RARQ---------------------QQQRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKA VVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPV STYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLH TSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIF NPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLY YVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNGGS AGSGHHHHHH(the the symbol “-” indicates that theamino acid at this position is deleted) RSV F furmt Truncated HIS(SEQ ID NO: 50) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCN GTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARKELPRFMNYTLNNAKKTNVTLSKKRKKKFLGF LLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQ SCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQI VRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVS FFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKT KCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDA SISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNGGSAGSGHHHHHH RSV F furdel Truncated HIS (SEQ ID NO: 51)MELLILKANAITTILTAVTFCFASGQNITEEFYQS TCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNN RARQELPRFMNYTLNNAKKTNVTLSKK---RFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTN KAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTT PVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWK LHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVD IFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNT LYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNG GSAGSGHHHHHH(the the symbol “-” idicates thatthe amino acid at this position is deleted)RSV F Factor Xa Truncated HIS (SEQ ID NO: 52)MELLILKANAITTILTAVTFCFASGQNITEEEYQS TCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNN IEGRELPRFMNYTLNNAKKTNVTLSKKIEGRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTN KAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTT PVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWK LHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVD IFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNT LYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNG GSAGSGHHHHHHRSV F Short linker Foldon HIS (SEQ ID NO: 53)MELLILKANAITTILTAVTFCFASGQNITEEFYQST CSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNR ARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNK AVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTP VSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKL HTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDI FNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTL YYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNGS GYIPEAPRDGQAYVRKDGEWVLLSTFLGGSAGSGHHHHHH RSV F Long linker Foldon HIS (SEQ ID NO: 54)MELLILKANAITTILTAVTFCFASGQNITEEFYQS TCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNN RARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTN KAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTT PVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWK LHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVD IFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNT LYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNN KNDDKGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSAGSGHHHHHH RSV_F_ecto_pre_his (SEQ ID NO: 55)MELLILKANAITTILTAVTFCFASGQNITEEFYQS TCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNN RARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTN KAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTT PVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWK LHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVD IFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNT LYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNDKIEEILS KIYHIENEIARIKKLIGESGGSAGSGHHHHHHECTO PRE HA HIS (SEQ ID NO: 56) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCN GTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGF LLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQ SCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQI VRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVS FFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKT KCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDA SISQVNEKINQSLAFIRKSDELLHNVNEKFHQIEKEFSEVEGRIQDLEKSGGSAGSGHHHHHH RSV F ECTO Furx GCN HIS (SEQ ID NO: 57)MELLILKANAITTILTAVTFCFASGQNITEEFYQS TCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNN QAQNELPQFMNYTLNNANNTNVTLSQNQNQNFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTN KAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTT PVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWK LHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVD IFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNT LYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNDKIEEILS KIYHIENEIARIKKLIGESGGSAGSGHHHHHHRSV F ECTO delp21 GCN HIS (SEQ ID NO: 58)MELLILKANAITTILTAVTFCFASGQNITEEFYQS TCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNN QAQN---------------------QNQNQNFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTN KAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTT PVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWK LHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVD IFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNT LYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNDKIEEILS KIYHIENEIARIKKLIGESGGSAGSGHHHHHH(the the symbol “-” idicates that theamino acid at this position is deleted) RSV F ECTO delp23 Furx GCN HIS(SEQ ID NO: 59) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCN GTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNQAQN-----------------------QNQNFLGF LLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQ SCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQI VRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVS FFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKT KCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDA SISQVNEKINQSLAFIRKSDELLHNVNDKIEEILSKIYHIENEIARIKKLIGESGGSAGSGHHHHHH (the symbol “-” idicates that the aminoacid at this position is deleted) RSV F ECTO delp23 Furdel GCN HIS(SEQ ID NO: 60) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCN GTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARQ-----------------------QQQRFLGF LLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQ SCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQI VRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVS FFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKT KCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDA SISQVNEKINQSLAFIRKSDELLHNVNDKIEEILSKIYHIENEIARIKKLIGESGGSAGSGHHHHHH (the symbol “-” idicates that the aminoacid at this position is deleted) RSV F Full Length Furx (SEQ ID NO: 61)MELLILKANAITTILTAVTFCFASGQNITEEFYQS TCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNN QAQNELPQFMNYTLNNANNTNVTLSQNQNQNFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTN KAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTT PVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWK LHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVD IFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNT LYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNI MITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN RSV F Full Length delp21 (SEQ ID NO: 62)MELLILKANAITTILTAVTFCFASGQNITEEFYQS TCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQL1MQSTPATNN QAQN---------------------QNQNQNFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTN KAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTT PVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWK LHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVD IFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNT LYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNI MITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN (the symbol “-” idicates that the aminoacid at this position is deleted) RSV F Full Length p23 Furx GCN HIS(SEQ ID NO: 63) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCN GTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNQAQN---------------------QNQNFLGFLL GVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSC SISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVR QQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFF PQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKC TASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASI SQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKD QLSGINNIAFSN(the symbol “-” idicates that the aminoacid at this position is deleted) RSV F Full Length p23 Furdel GCN HIS(SEQ ID NO: 64) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCN GTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARQ-----------------------QQQRFLGF LLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQ SCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQI VRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVS FFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKT KCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDA SISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLS KDQLSGINNIAFSN(the symbol “-” idicates that the aminoacid at this position is deleted) RSV F N-term Furin Furx Truncated HIS(SEQ ID NO: 65) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCN GTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNQAQNELPQFMNYTLNNAQQTNVTLSKKRKRRFLGF LLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQ SCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQI VRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVS FFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKT KCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDA SISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNGGSAGSGHHHHHH RSV F C-term Furin Furx Truncated HIS (SEQ ID NO: 66)MELLILKANAITTILTAVTFCFASGQNITEEEYQS TCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNN RARRELPQFMNYTLNNAQQTNVTLSQNQNQNFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTN KAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTT PVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWK LHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVD IFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNT LYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNG GSAGSGHHHHHHRSV F Fusion Deletion 1 Truncated HIS (SEQ ID NO: 67)MELLILKANAITTILTAVTFCFASGQNITEEFYQS TCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNN RARRELPRFMNYTLNNAKKTNVTLSKKRKRRSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNG VSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTN SELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTT NTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCK IMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEG KSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNGGSAGSGHHH HHHRSV F Fusion Deletion 2 Truncated HIS (SEQ ID NO: 68)MELLILKANAITTILTAVTFCFASGQNITEEFYQS TCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNN RARRELPRFMNYTLNNAKKTNVTLSKKRKRRGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSL SNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYM LTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPL CTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKY DCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNK QEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNGGSAGSG HHHHHH RSV F Furx Truncated HIS(SEQ ID NO: 69) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCN GTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNQAQNELPQFMNYTLNNAQQTNVTLSQNQNQNFLGF LLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQ SCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQI VRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVS FFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKT KCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDA SISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNGGSAGSGHHHHHH RSV F Furx Truncated (SEQ ID NO: 70)MELLILKANAITTILTAVTFCFASGQNITEEFYQS TCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNN QAQNELPQFMNYTLNNAQQTNVTLSQNQNQNFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTN KAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTT PVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWK LHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVD IFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNT LYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTN RSV F delP23 furdel Truncated No HIS(For CHO cells) (SEQ ID NO: 71) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCN GTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARQ-----------------------QQQRFLGF LLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQ SCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQI VRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVS FFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKT KCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDA SISQVNEKINQSLAFIRKSDELLHNVNAGKSTTN(the symbol “-” idicates that the aminoacid at this position is deleted) RSV F (Wt) Truncated HIS(SEQ ID NO: 84) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCN GTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGF LLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQ SCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQI VRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVS FFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKT KCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDA SISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNGGSAGSGHHHHHH RSV F old furx Truncated HIS (SEQ ID NO: 88)MELLILKANAITTILTAVTFCFASGQNITEEFYQS TCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNN QAQNELQRFMNYTLNNANNTNVTLSQNQNQNFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTN KAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTT PVSTYMLTNSELLSLINDMPITNDQKKLMSNNVOIVROOSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWK LHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSEFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVD IFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNT LYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNG GSAGSGHHHHHHRSV F Furx R113Q K123N K124N Truncated HIS (SEQ ID NO: 89)MELLILKANAITTILTAVTFCFASGQNITEEFYQS TCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNN QAQNELPQFMNYTLNNAQQTNVTLSQNQNQNFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTN KAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTT PVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWK LHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVD IFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNT LYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNG GSAGSGHHHHHHRSV F N-term Furin Truncated HIS (SEQ ID NO: 85)MELLILKANAITTILTAVTFCFASGQNITEEFYQS TCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNN RARRELPQFMNYTLNNAQQTNVTLSQNQNQNFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTN KAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTT PVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWK LHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVD IFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNT LYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNG GSAGSGHHHHHHRSV F delP21 furdel Truncated HIS (SEQ ID NO: 86)MELLILKANAITTILTAVTFCFASGQNITEEFYQS TCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNN RARQ---------------------QNQQQRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTN KAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTT PVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWK LHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVD IFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNT LYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNG GSAGSGHHHHHHRSV F C-term Furin Truncated HIS (SEQ ID NO: 87)MELLILKANAITTILTAVTFCFASGQNITEEEYQS TCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNN QAQNELPQFMNYTLNNAQQTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTN KAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTT PVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWK LHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVD IFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNT LYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNG GSAGSGHHHHHH

Example 2—Expression and Purification of RSV F Constructs

The RSV F ECTO and truncated constructs, lacking the transmembranedomain and cytoplasmic tail region with either wild-type furin cleavagesites or harboring knock-out mutations to the furin cleavage sites andwith or without prefusion stabilization mutations, were cloned into apFastBac baculovirus expression vector (Invitrogen). Several of theseconstructs contain a C-terminal flexible linker followed by a His6-tagsequence used for chelating purification. The production of high-titerbaculovirus stocks were passaged in Sf9 insect cells. Proteins wereexpressed by infecting either Sf9, Tn5 or High Five insect cells withthe required baculovirus and harvesting the media supernatant two orthree days post infection, monitored by western blot using an anti-RSV For anti-6HIS antibody.

Large scale expression media was concentrated/purified by one of twogeneral strategies for eliminating the deleterious Effect of theferritin present in insect cell media from corrupting the chelatingresin. The first approach was to concentrate the approximately 10-20liters of insect expression media down to approximately 300 mls using aGE Healthcare Hollotube fiber concentration column. Copper sulfate wasadded to this concentrated mixture to a final concentration of 500 μMand the resulting solution was loaded onto 5 ml HiTrap chelatingcolumns. The bound HIS-tagged protein was then eluted from the columnwith 25 mM Tris pH 7.5, 300 mM NaCl and a gradient of imidazole.

In the second purification strategy, CuCl₂ was added to mediasupernatant to a final concentration of 500 μM. To each 1 liter ofmedia, four milliliters of chelation resin (Chelating Resin, BioRad) wasadded and the slurry was rocked for at least thirty minutes at 4 degreescentigrade and the resin and media were separated by a gravity column.The resin was washed with ten-times column volume of equilibrationbuffer (25 mM Tris pH 7.5, 300 mM NaCl) and the F protein was elutedwith ten-times column volume of elution buffer (equilibration bufferwith 250 mM imidazole). The elution was dialyzed against 25 mM Trisbuffer pH 7.5, and the resulting solution was loaded onto a 5 ml Hitrapchelation column charged with NiSO4 and eluted with 25 mM Tris pH 7.5,300 mM NaCl and a gradient of imidazole.

Elutions from the imidazole gradient in either case were evaluated usinganti-6HIS western and coomassie gels. Fractions containing pureconstructs were collected, dialyzed against different buffer/salinesolutions and were concentrated for subsequent analysis using MilliporeCentriprep Concentrators and/or Vivaspin concentration units. We havealso developed a size-exclusion purification protocol capable of furtherpurifying monodisperced RSV trimers from rosettes (below).

SEC Analysis of RSV F Proteins:

A documented feature of other paramyxovirus fusion proteins stabilizedin their prefusion conformation is that, even when cleaved so that thefusion peptide is exposed, they do not form rosettes as is observed forthe postfusion conformation. A simple size-exclusion chromatographyanalysis allows for identification of a protein and determination ofwhether a protein is forming rosettes. Two methods were developed,HPLC-SEC and FPLC-SEC, which also serves as an efficient purificationstep.

HPLC-SEC was performed using a Biorad SEC column (18 mm) with a 25 mMTris pH 7.5, 300 mM NaCl mobile phase. Using Biorad HPLC-SEC standardsto calibrate the system, we found that the RSV rosettes (representingcleaved-postfusion conformations) elute in the column void volume of theanalysis, while RSV monodispersed trimers (presumed trimers fromsubsequent EM analysis) elute with an apparent molecular weight ofapproximately 100 kDa.

FPLC-SEC was performed on a GE Healtcare FPLC using a 16/60 Superdex 200column with 25 mM Tris pH 7.5, 300 mM NaCl mobile phase. Using GEHealthcare High molecular weight standards to calibrate the system, wefound that the RSV rosettes elute in the column void volume of theanalysis, while RSV monodispersed trimers elute with an apparentmolecular weight of approximately 100 kDa.

Electron Microscopy (EM) of RSV F Proteins.

Protein solutions of approximately 50 micrograms per ml RSV F constructswere absorbed onto glow-discharged carbon coated grids and werenegatively stained with 2% sodium phosphotungstate (pH 7.0) or 0.75%Urynal-formate (unquantified low pH). The grids were observed on aTechnai Spirit or JOEL 1230 transmission electron microscope operatingbetween 80-120 kV with a magnification between 20,000 to 150,000depending on required resolution.

TABLE 2 Construct Conformation by EM RSV F ECTO HIS Predominatelyrosettes RSV F Furdel ECTO (cleaved) Predominately rosettes RSV F Delp23Furdel Truncated Trimers observed (uncleaved) RSV F Fusion PeptideDeletion 1 Timers Truncated (uncleaved) RSV F Delp23 Furdel TruncatedPredominantly rosettes (cleaved by Trypsin after purification) with sometrimers RSV F Delp23 Furdel Truncated Asymmetric rosettes with (cleavedby Trypsin after purification apparent nanolipid disk at in presence ofnanolipid disk) the center of rosette

Example 3—Detection of Pre-Fusion and Post-Fusion RSV F

A number of methods are available to determine the conformation of theRSV F protein to assay whether a modification to the RSV F polypeptideor added molecule disfavors the post-fusion conformation. Examplesinclude liposome association, conformation specific monoclonalantibodies (including as used in FACS, ELISA, etc.), electronmicroscopy, differential protease sensitivity between the conformations,gel filtration chromatography, analytical ultracentrifugation, dynamiclight scattering, deuterium exchange NMR experiments, mass spectroscopy,circular dichroism spectroscopy, isothermal titration calorimetry,tryptophan spectroscopy, and X-ray crystallography.

Liposome Association

Liposome association may be used to assay the conformation of the RSV Fprotein. Soluble forms of the RSV F protein in the pre-fusionconformation will not associate with liposomes while the post-fusionconformation will associate with liposomes.

Liposomes may be prepared as follows:1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine,1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, and cholesterolin chloroform (available from Avanti Polar Lipids) are mixed at an 8:2:5molar ratio. The chloroform is evaporated under argon. A lipid film willform that is dried under vacuum overnight and resuspended in PBS at 40mM total lipid. After five freeze-thaw cycles, the lipids are vortexedand extruded 21 times through two 100-μm filters by using a miniextruder(available from Avanti Polar Lipids).

Once the liposomes have been prepared, the liposome association assaymay be performed. For each sample to be tested, 2 μg of the RSV Fpolypeptide to test is cleaved with 25 milliunits of trypsin (availablefrom Worthington Biochemical) in 100 mM phosphate buffer (pH 7.1) for 30min at 25° C. After cleavage, 40 μg of soybean trypsin inhibitor(available from Worthington Biochemical) is added to each sample to endthe reaction. The samples are pretreated at 60° C. for 30 min whichwould induce a conformational shift from the pre-fusion to thepost-fusion forms in native isolated RSF F protein. Liposomes (40 μl persample) and PBS are added (80 μl final volume), and the samples areincubated at 60° C. for 30 min. Sucrose is added to a finalconcentration of 50% (500 μl final volume). The samples are overlaidwith 500 μl each of 40% sucrose, 25% sucrose, and PBS and are spun in aTLS55 rotor at 49,000 rpm for 3 h at 25° C. Fractions (500 μl) arecollected from the top of the gradients. Proteins are solubilized in0.5% Triton X-100 and precipitated by using 12.5% vol/voltrichloroacetic acid. Polypeptides are separated by SDS/PAGE andtransferred to PVDF membranes. Blots are probed with anti-RSV Fmonoclonal antibodies.

Electron Microscopy

Electron microscopy was used to assay the conformational distribution ofRSV F polypeptides. RSV F polypeptides in the pre-fusion form have a“ball and stem” shape with a length of ˜12 nm. In contrast, RSV Fpolypeptides in the post-fusion form have a “golf tee” shape with alength of ˜16 nm. In addition, the fusion peptides at the narrow end ofthe “golf tees” aggregate to form a rosette structures. Thus, electronmicroscopy may be used to assay the distribution of conformations in asample of RSV F polypeptides owing to the readily distinguishableshapes.

Example 4 RSV F Ectodomain Trimers and Rosettes

The RSV F protein ecto-domain constructs, encoding polypeptides thatlack the transmembrane domain and cytoplasmic tail region with eitherwild-type furin cleavage sites or harboring knock-out mutations to thefurin cleavage sites and/or fusion peptide mutations mutations, werecloned into a pFastBac baculovirus expression vector (Invitrogen).Several of these constructs contain a C-terminal flexible linkerfollowed by a HIS6-tag sequence used for chelating purification. Theproduction of high-titer baculovirus stocks were obtained by passage inSf9 insect cells. Proteins were expressed by infecting either Sf9, Tn5or High Five insect cells with the required baculovirus and harvestingthe conditioned media supernatant two or three days post infection.Protein production was monitored by western blot using an anti-RSV F oranti-HIS6 antibody.

Large scale expression media was concentrated/purified using one of twogeneral strategies for eliminating the deleterious effect of theferritin present in insect cell media, which can corrupt the chelatingresin. The first approach was to concentrate the approximately 10-20liters of insect expression media down to approximately 300 mls using aGE Healthcare Hollotube fiber concentration column. Copper sulfate wasadded to this concentrated mixture to a final concentration of 500 μM,and the resulting solution was loaded onto 5 ml HiTrap chelatingcolumns. The bound HIS-tagged protein was then eluted from the columnwith 25 mM Tris pH 7.5, 300 mM NaCl and a gradient of imidazole.

In the second purification strategy, CuCl₂ was added to mediasupernatant to a final concentration of 500 μM. To each 1 liter ofmedia, approximately four to ten milliliters of chelating resin(Chelating Resin, BioRad) was added, and the slurry was rocked for atleast thirty minutes at 4 degrees centigrade, and the resin and mediawere separated using a gravity column. The resin was washed withapproximately ten times the column volume of equilibration buffer (25 mMTris pH 7.5, 300 mM NaCl), and the F protein ecto-domain was eluted withapproximately ten-times the column volume of elution buffer(equilibration buffer with 250 mM imidazole). The elution was dialyzedagainst 25 mM Tris buffer pH 7.5, and the resulting solution was loadedonto a 5 ml Hitrap chelation column charged with NiSO4. Bound proteinwas eluted with 25 mM Tris pH 7.5, 300 mM NaCl and a gradient ofimidazole.

Elutions from the imidazole gradient in either case were evaluated usinganti-HIS6 western blots and/or Coomassie-stained SDS-PAGE gels.Fractions containing pure constructs were collected, dialyzed againstdifferent buffer/saline solutions and were concentrated for subsequentanalysis using Millipore Centriprep concentrators and/or Vivaspinconcentration units. In some instances, monomers, trimers or rosetteswere further purified using size exclusion chromatography.

SEC Analysis and Purification of RSV F Ecto-Domains

Size exclusion chromatography was used to purify and analyze RSV Fprotein ecto-domain monomers, trimers and rosettes. This method alsoallowed uncleaved RSV F protein ecto-domains to be purified away fromhost cell or media derived lipid and lipoprotein contaminants. Twomethods were developed, HPLC-SEC and FPLC-SEC, which may also serve asan efficient purification step.

HPLC-SEC was performed using a Biorad SEC column (18 mm) with a 25 mMTris pH 7.5, 300 mM NaCl mobile phase. Using Biorad HPLC-SEC standardsto calibrate the system, we found that the RSV rosettes (representingcleaved, postfusion conformations) elute in the column void volume ofthe analysis, while RSV F monomers elute with an apparent molecularweight of approximately 75-85 kDa.

FPLC-SEC was performed on a GE Healthcare FPLC using a 16/60 Superdex200 column with 25 mM Tris pH 7.5, 300 mM NaCl as a mobile phase. UsingGE Healthcare High molecular weight standards to calibrate the system,we found that the RSV rosettes elute in the column void volume of theanalysis, while RSV monodispersed trimers elute with an apparentmolecular weight of approximately 140-160 kDa and RSV F monomers elutewith an apparent molecular weight of approximately 75-85 kDa.

For purification, the FPLC-SEC method was used and 1 ml fractions werecollected.

Typsin Cleavage of Furdel or Delp23 Furdel Constructs to Form PostfusionRosettes

In general, trypsin digestion of Delp23 Furdel monomers is done with1:1000 trypsin:RSV F by weight, or 10-15 BAEE units of trypsin for 1 mgof RSV F antigen. In a typical reaction, trypsin from bovine plasma(Sigma Aldrich, T8802: 10,000-15,000 BAEE units/mg trypsin) was dilutedto a 1 mg/ml concentration in 25 mM Tris pH 7.5, 300 mM NaCl. A 1 mg/mlsolution of RSV F protein ecto-domain polypeptide (diluted in 25 mM TrispH 7.5, 300 mM NaCl) was treated with one microliter of trypsin solution(final mass ratio 0.001:1 trypsin:RSV F or approximately 10-15 BAEEunits of trypsin to each milligram of RSV F) for 1 hour at 37° C.Typically, progress of the cleavage reaction was monitored by SDS-PAGEgel. The cleavage reaction was stopped using a trypsin inhibitor. Thecleaved RSV F protein was further purified by size exclusionchromatography.

On occasion, 1:100 volume of immobilized trypsin inhibitor (Sigma) or 1microliter of 1 mM soybean trypsin inhibitor was added to the cleavagesolution and the mixture was incubated at room temperature forapproximately 15-30 minutes with gentle rocking to stop the trypsinreaction. The inhibitor resin was separated from the protein solutionusing microcentrifuge columns. The resulting solution was purified bySEC purification.

Electron Microscopy (EM) of RSV F Proteins.

RSV F protein ecto-domain polypeptides (approximately 50 micrograms perml), were absorbed onto glow-discharged carbon-coated grids and werenegatively stained with 2% sodium phosphotungstate (pH 7.0) or 0.75%uranyl-formate (unquantified low pH). The grids were observed on aTechnai Spirit or JOEL 1230 transmission electron microscope operatingbetween 80-120 kV with a magnification between 20,000 to 150,000depending on required resolution.

Phospholipid Assay

This assay is based on the Wako Pure Chemical Industries, Ltd. assayPhospholipids C choline oxidase—DAOS method (Cat. No 433-36201). Theassay protocol is modified only to reduce the amount of material used inthe assay, and to decrease the sample dilution in the reaction relativeto the general protocol from the vendor. To determine the lipid contentof the RSV F sample, generate the color reagent by dissolving one bottleof Color Reagent with one bottle of Buffer (color reagent is stable for1 week at 4° C.). Dilute the 300 mg/dL (3 mg/ml) phospholipid standardto 1.5, 1.0, 0.75, 0.5 and 0.25 mg/ml with distilled water. For eachstandard, a water blank and sample reaction, add to a microcentrifugetube 10 μl of color reagent and 2 μl of either standard, distilled water(0 mg/ml standard) or sample. Centrifuge reactions briefly to ensureproper mixing and incubate the tubes for 15 min at 37° C. Record theabsorbance at 595 nm for each standard point and generate a standardcurve. Record the 595 nm absorbance for each sample and calculate thephospholipids concentration from the prepared calibration curve.

Immunogenicity in Cotton Rats

Immunogenicity of RSV F protein ectodomain polypeptides in the forms ofmonomers (uncleaved delp21 furx), rosettes of trimers (cleaved delp23furdel), and trimers (fusion peptide deletion) were determined in cottonrats (Sigmodon hispidus) in two studies. In study 1 (FIGS. 8A and 8B),10 cotton rats per group were vaccinated intramuscularly with 10 μg ofmonomers or rosettes (each adsorbed to aluminum hydroxide) on days 0 and21. Serum anti-RSV F protein IgG and RSV neutralizing antibody titerswere measured 2 weeks after the 1^(st) vaccination (2wp1) and 2 weeksafter the 2^(nd) vaccination (2wp2), or 3 weeks after the 1^(st)vaccination (3wp1) and 2 weeks after the 2^(nd) vaccination (2wp2).Anti-RSV F protein IgG (FIG. 8A) was determined by ELISA using RSV Fprotein coated plates and horse radish peroxidase-conjugated chickenanti-cotton rat IgG detection antibody. Data are presented as log₁₀geometric mean titers (GMT)+standard deviations of individual cottonrats. RSV neutralization titers (FIG. 8B) were measured by plaquereduction neutralization test (PRNT). In brief, dilutions ofheat-inactivated serum were preincubated with RSV Long, and theninoculated on HEp-2 cells in 12-well plates. After a 2 hour infectionthe inoculum was removed and cells were overlaid with agarose. Plaqueswere enumerated 5 days later by neutral red staining. The neutralizationtiter is defined as the reciprocal of the serum dilution producing atleast a 60% reduction in number of plaques per well, relative tocontrols (no serum). Data are presented as log₁₀ GMT+standard error of 2pools of 5 cotton rats per group.

In study 2 (FIG. 8C), 9 cotton rats per group were immunizedintramuscularly with the indicated doses of monomers, trimers, orrosettes (each adsorbed to aluminum hydroxide). Serum anti-RSV F proteinIgG titers were measured 2wp1 as above.

Results

The soluble RSV F ecto-domain (with un-mutated furin cleavage sites) wasexpressed, but could not be purified from lipid and lipoproteinimpurities derived from the host cells or culture media using sizeexclusion chromatography. These RSV F ecto-domain polypeptides elute inthe SEC column void volume along with the lipid and lipoproteincontaminants.

Several constructs were prepared for making RSV F ecto-domainpolypeptides that contain mutations to the furin cleavage sites,including the Furdel constructs. See, FIGS. 1A-1C. Polypeptides producedby expression of the Furdel constructs were secreted from the cells asan ˜65 kDa uncleaved species. The Furdel mutation also prevents fusionpeptide exposure, which in turn prevents rosette formation. As a result,soluble RSV F Furdel migrated in the included volume of a Superdex 200preparatory column, resulting in separation from both the lipid debris,which eluted in the void volume, and insect protein impurity. Theseresults show that RSV F ecto-domain polypeptides in which the furincleavage sites have been mutated are produced as uncleaved polypeptidethat can be purified by SEC. In addition, analysis of the uncleaved RSVF retention time was consistent with the polypeptides being monomersrather than trimers.

Whether the RSV F furdel polypeptides were monomers, trimers or amixture of monomers and trimers was assessed further using analyticalultracentrifugation. Analytical ultracentrifugation studies wereperformed using protein purified from the monomer peak from the SECpurification. Sedimentation velocity data of the uncleaved RSV F showeda step pattern suggesting two species in solution. Analysis of thesedimentation velocity experiment showed that the uncleaved RSV Fecto-domain had a high population of monomer and a minor population ofapparent trimer in the solution. Equilibrium run data was collected andattempts to fit the data to either an ideal monomer model or amonomer-trimer equilibrium model were performed. However, the residualsof the fits are poor, particularly toward the bottom of the cell wherethe protein concentration is higher. These observations suggested thatthe uncleaved RSV F ecto-domain polypeptides are predominantly a monomerwith a smaller population which self associates (potentially as trimers)or aggregates at higher concentrations.

Further analysis of select RSV F protein ectodomain polypeptides wasconducted using size exclusion (SEC) chromatography. FIGS. 6A-6D. Theprinciple peaks containing monomers, trimers or rosettes of trimers areindicated by an asterisk in FIGS. 6A-6D, with the retention time of theSuperdex P200 16/60 column (GE Healthcare) is indicated in milliliters.On a calibrated column, the approximate retention times of 47 mls, 65mls and 77 mls correspond to the column void volume, the retention of Ftrimers and the retention of the monomers, respectively. In FIG. 6A, theuncleaved Delp23 Furdel (Δp23 Furdel) construct was purified from themonomer peak. When the uncleaved Delp23 Furdel RSV F antigen was treatedwith trypsin, the protein formed rosettes, which migrated on SEC in thevoid volume (FIG. 6B). The cleaved trimer species of RSV F fusionpeptide deletion was purified from the trimer peak at approximately 65mls retention time (FIG. 6C) while the uncleaved Delp21 Furx construct(Δp21 Furx) was purified from the monomer peak at approximately 77 mls(FIG. 6D).

Several RSV F protein ecto-domain polypeptides in uncleaved form orafter trypsin cleavage were assessed by EM. The RSV F Furdel and delp23Furdel constructs have arginine residues remaining in the furin cleavagesite. These arginines are susceptible to trypsin cleavage. Uponcleavage, the uncleaved F₀ species was converted to the F₁/F₂ species,in which the fusion peptide is exposed. EM analysis confirmed thatfollowing trypsin cleavage the uncleaved RSV ecto-domains formedrosettes of trimers by virtue of their fusion peptides, as has beenobserved for related fusion proteins. The results are presented in theTable 3, and show that uncleaved RSV F protein ecto-domain polypeptidescan be cleaved to form rosettes of trimers. The fusion peptide deletedconstruct, which is cleaved by furin, formed monodispersed trimers. See,also, FIGS. 7A-7D. Advantageously, producing rosettes of trimers in thisway results in rosettes of trimers that are substantially free of lipiddebris and lipoproteins.

The results of the immunogenicity studies showed that RSV F proteinectodomain polypeptides in the form of monomers (uncleaved delp2 furx),rosettes of trimers (cleaved delp23 furdel), and trimers (fusion peptidedeletion) were immunogenic in cotton rats (Sigmodon hispidus), andinduced neutralizing antibodies. FIG. 8A-8C.

TABLE 3 Construct Conformation by EM RSV F wild type ecto-domainRosettes of trimers associated (cleaved in host cell during with lipiddebris expression) Trypsin-cleavable Furdel (purified Variable. Somepreparations show monomer peak - uncleaved) monodispersed trimers;others show little material visible by EM of negatively-stained materialTrypsin-cleavable Furdel (purified Rosettes of trimers monomer peak -trypsin cleaved after purification) Trypsin-cleavable delp23 furdelVariable. Some preparations show (purified monomer peak - uncleaved)monodispersed trimers; others show little material visible by EM ofnegatively-stained material Trypsin-cleavable delp23 Furdel Rosettes oftrimers (purified monomer peak - trypsin cleaved after purification)Cleaved Fusion Peptide Deletion Monodispersed trimers (purified monomerpeak)

Example 5—Methods for Making RSV F Subunit Antigens in Insect or CHOCells

RSV F Antigen Purification from Insect Cells:

RSV F ectodomain subunits, including Delp21 Furx, Delp23 Furdel andFusion Peptide Deletion constructs, were expressed in HiFive insectcells (Invitrogen) using the pFAST Bac baculovirus system. The RSV Fsubunit was purified from large scale expressions, 10-25 liters, via atwo step chelating method that reduced the deleterious effect of theferritin containment present in insect cell media, which can corrupt thechelating resin. CuSO₄ was added to media supernatant to a finalconcentration of 500 μM. Approximately ten to twenty milliliters ofchelating resin (Chelating Resin, BioRad) was added to each 1 liter ofmedia, the slurry was rocked for at least thirty minutes at 4° C., andthe resin and media were separated using a gravity column. The resin waswashed with approximately two-times the resin volume of equilibrationbuffer (25 mM Tris pH 7.5, 300 mM NaCl), and the F protein ecto-domainwas eluted with approximately two-times the column volume of elutionbuffer (equilibration buffer with 250 mM imidazole). The elution wasdialyzed against 25 mM Tris buffer pH 7.5, 300 mM NaCl and the resultingsolution was loaded onto a 5 ml Hitrap chelation column charged withNiSO4 (GE Healthcare). Bound protein was eluted with 25 mM Tris pH 7.5,300 mM NaCl and a gradient of imidazole.

Elutions from the imidazole gradient in both cases were evaluated usinganti-HIS6 western blots and/or Coomassie-stained SDS-PAGE gels.Fractions containing pure constructs were collected and concentrated toapproximately 1 mg/ml using Millipore Centriprep concentrators and/orVivaspin concentration units for subsequent analysis/purification bysize exclusion chomatography.

SEC Analysis and Purification of RSV F Ectodomains

Size exclusion chromatography (SEC) was used to purify and analyze RSV Fprotein ectodomain uncleaved monomers and cleaved trimers. This methodalso allowed uncleaved RSV F protein ectodomains to be purified awayfrom host cell or media derived lipid and lipoprotein contaminants. Inthe case of clean-rosette generation, the uncleaved Delp23 Furdelconstruct was initially purified as a monomer and subsequently proteasetreated and re-purified using SEC to purify homogeneous rosettes (seebelow). Two methods were developed for analysis of RSV Foligamerization, HPLC-SEC and FPLC-SEC, which may also serve as anefficient purification step.

HPLC-SEC was performed using a Biorad SEC column (18 mm) with a 25 mMTris pH 7.5, 300 mM NaCl mobile phase. Using Biorad HPLC-SEC standardsto calibrate the system, we found that the RSV rosettes (representingcleaved, postfusion conformations) elute in the column void volume ofthe analysis while RSV F monomers elute with an apparent molecularweight of approximately 75-85 kDa.

FPLC-SEC was performed on a GE Healthcare FPLC using a 16/60 Superdex200 column with 25 mM Tris pH 7.5, 300 mM NaCl as a mobile phase. UsingGE Healthcare High molecular weight standards to calibrate the system,we found that the RSV rosettes elute in the column void volume of theanalysis, while RSV monodispersed trimers elute with an apparentmolecular weight of approximately 140-160 kDa and RSV F monomers elutewith an apparent molecular weight of approximately 75-85 kDa. Forpurification of RSV uncleaved Delp21 Furx or Delp23 Furdel (monomers) orFusion Peptide Deletion (trimer) 0.5-2 mls of approximately 1 mg/mlchelation purified material was loaded on to an equilibrated SuperdexP200 16/60 column with a flow rate between 0.5-2 mls/min and relevantfractions were collected.

Typsin Cleavage of Delp23 Furdel Constructs to Form Postfusion Rosettes

Trypsin from bovine plasma (Sigma Aldrich, T8802: 10,000-15,000 BAEEunits/mg trypsin) was diluted to a 1 mg/ml concentration in 25 mM TrispH 7.5, 300 mM NaCl. A 1 mg/ml solution of RSV F protein ecto-domainpolypeptide (diluted in 25 mM Tris pH 7.5, 300 mM NaCl) was treated withone microliter of trypsin solution (final mass ratio 0.001:1 trypsin:RSVF or approximately 10-15 BAEE units of trypsin to each milligram of RSVF) for 1 hour at 37° C. Progress of the cleavage reaction was monitoredby SDS-PAGE gel. The cleavage reaction was stopped using a trypsininhibitor (Gibco Soy Bean Trypsin Inhibitor using equal mass ofinhibitor to trypsin). It was found that an incubation period wasrequired between the cleavage step and subsequent rosette purificationto allow higher efficiency of monomer to rosette conversion. A one to 6hour incubation period at 37° C. was given to provide higher rosetteformation efficiency. The cleaved RSV F protein was further purifiedfrom unconverted monomer species using size exclusion chromatography (asdescribed above) where homogeneous rosettes can be collected in thecolumn void volume fractions.

RSV F Antigen Purification from CHO Cells:

RSV F Fusion Peptide Deletion constructs, which do not contain aHIS-tag, were purified by cation purification. CHO material containingexpressed RSV F trimer antigen was concentrated to approximatelyone-tenth the original volume on a GE Healthcare hollow fiber cartridgeconcentration system (MWCO 10,000 kDa). The concentrated solution wasthen buffer exchanged four times with an equivalent volume of 25 mMSodium Acetate pH 6.0, 25 mM NaCl. The resulting solution, containingconcentrated RSV F trimer in the acetate/saline buffer, was loaded ontoa pre-charged GE Healthcare HiTrap CM column which had been equilibratedwith acetate/saline buffer. The protein was eluted from the column usinga step gradient of 25 mM acetate buffer containing either 25, 150, 250,500 or 1000 mM NaCl (the 250 mM and 500 mM NaCl fractions containing thebulk of the eluted material). This material could be further purifiedusing a SEC purification similar to the protocol above.

Example 6—Immunogenicity of RSV F Subunits in Cotton Rats

The immunogenicity and protective capacity of RSV-F trimer(RSV-F-fusion-peptide-deletion-trun) and rosette(RSV-F-delp23-furdel-trunc,cleaved) subunits, each formulated with alumor MF59, was evaluated in the cotton rat model. The antigen used forELISA in this study was RSV-F-fusion-peptide-deletion-trunc (Table 4).Neutralization was against infectious RSV, strain Long (Table 5). Allcombinations were immunogenic, eliciting high titer RSV-F-specific IgGand RSV neutralizing antibody responses that were boosted by a secondvaccination, and afforded protection from nasal RSV challenge.

Methods

Vaccination and Challenge of Cotton Rats

Female cotton rats (Sigmodon hispidis) were obtained from HarlanLaboratories. Groups of animals were immunized intramuscularly (i.m.,100 μl) with the indicated vaccines on days 0 and 21. Serum samples werecollected 3 weeks after the first immunization (3wp1) and 2 weeks afterthe second immunization (2wp2). Immunized or unvaccinated controlanimals were challenged intranasally (i.n.) with 1×10⁵ pfu RSV Long 4weeks after the final immunization. Blood collection and RSV challengewere performed under anesthesia with 3% isoflurane using a precisionvaporizer.

RSV F-Specific ELISA

Individual serum samples were assayed for the presence of RSV F-specificIgG by enzyme-linked immunosorbent assay (ELISA). ELISA plates (MaxiSorp96-well, Nunc) were coated overnight at 4° C. with 1 μg/ml purified RSVF (fusion-peptide deletion-trunc) in PBS. After washing (PBS with 0.1%Tween-20), plates were blocked with Superblock Blocking Buffer in PBS(Thermo Scientific) for at least 1.5 hours at 37° C. The plates werethen washed, serial dilutions of serum in assay diluent (PBS with 0.1%Tween-20 and 5% goat serum) from experimental or control cotton ratswere added, and plates were incubated for 2 hours at 37° C. Afterwashing, plates were incubated with horse radish peroxidase(HRP)-conjugated chicken anti-cotton rat IgG (Immunology ConsultantsLaboratory, Inc, diluted 1:5,000 in assay diluent) for 1 hour at 37° C.Finally, plates were washed and 100 μl of TMB peroxidase substratesolution (Kirkegaard & Perry Laboratories, Inc) was added to each well.Reactions were stopped by addition of 100 μl of 1M H₃PO₄, and absorbancewas read at 450 nm using a plate reader. For each serum sample, a plotof optical density (OD) versus logarithm of the reciprocal serumdilution was generated by nonlinear regression (GraphPad Prism). Titerswere defined as the reciprocal serum dilution at an OD of approximately0.5 (normalized to a standard, pooled sera from RSV-infected cotton ratswith a defined titer of 1:2500, that was included on every plate).

Micro Neutralization Assay

Serum samples were tested for the presence of neutralizing antibodies bymicroneutralization assay. Two-fold serial dilutions of heat inactivated(HI)-serum (in PBS with 5% HI-fetal bovine serum(FBS)) were added to anequal volume of RSV, strain Long previously titered to giveapproximately 115 PFU/25 μl. Serum/virus mixtures were incubated for 2hours at 37° C. and 5% CO2, to allow virus neutralization to occur, andthen 25 μl of this mixture (containing approximately 115 PFU) wasinoculated on duplicate wells of HEp-2 cells in 96 well plates. After 2hours at 370 and 5% CO2, the cells were overlayed with 0.75% MethylCellulose/EMEM 5% HI-FBS and incubated for 42 hours. The number ofinfectious virus particles was determined by detection of syncytiaformation by immunostaining followed by automated counting. Theneutralization titer is defined as the reciprocal of the serum dilutionproducing at least a 60% reduction in number of synctia per well,relative to controls (no serum).

Viral Load

Viral load in the lung was determined by plaque assay. Specifically,lungs were harvested 5 days post RSV infection and one right lobe wasplaced into 2.5 ml Dulbecco's Modified Eagle Medium (DMEM, Invitrogen)with 25% sucrose and disrupted with a tissue homogenizer. Cell-freesupernatants from these samples were stored at −80° C. To assay forinfectious virus, dilutions of clarified lung homogenate (in PBS with 5%HI-FBS) were inoculated on confluent HEp-2 cell monolayers in a volumeof 200 μl/well of a 12-well plate. After 2 hours with periodic gentlerocking (37° C., 5% CO₂), the inoculum was removed, and cells wereoverlaid with 1.5 ml of 1.25% SeaPlaque agarose (Lonza) in Eagle'sMinimal Essential Medium (EMEM, Lonza) supplemented with 5% HI-FBS,glutamine, and antibiotics. After 3-4 days of incubation, cells wereagain overlaid with 1 ml of 1.25% agarose in EMEM (Sigma) containing0.10% neutral red (Sigma). Plaques were counted one day later with theaid of a light box.

An alternative method for determining viral load is quantitativereal-time PCR (qRT-PCR). Viral load can be determined by qRT-PCR usingoligonucleotide primers specific for the RSV-F gene as described (I.Borg et al, Eur Respir J 2003; 21:944-51) with some modifications.Briefly, RNA is isolated from 140 μl of clarified lung homogenate, orfrom a known number of plaque forming units (PFU) of RSV (determined byplaque assay, and diluted in lung homogenate from uninfected animals),using the RNeasy kit (Qiagen) with a final elution volume of 100 μl H₂O.cDNA synthesis and PCR is performed in a single tube using theSuperScript III Platinum One-Step Quantitative RT-PCR kit (Invitrogen)with 5 μL of eluted RNA, 10 μM of each primer, and 50 μM of the probe(primers and probes from Integrated DNA Technologies). Forward primer:TTGGATCTGCAATCGCCA (SEQ ID NO:72). Reverse primer:CTTTTGATCTTGTTCACTTCTCCTTCT (SEQ ID NO:73). Probe: 5′-carboxyfluorescein(FAM)-TGGCACTGCTGTATCTAAGGTCCTGCACT-tetramethylcarboxyrhodamine(TAMRA)-3′(SEQ ID NO:74). Amplification and detection is performed with an ABIPrism 7900HT or 7500 (Applied Biosystems). A threshold cycle value (Ct)is defined for each sample as the cycle number at which the fluorescentsignal first becomes detectable above a set threshold. PFU equivalentsfor each sample is then determined based on a standard curve of Ctverses the logarithm of defined copy number of viral RNA.

Results

The cotton rat as a model has been used extensively in the study of RSVpathogenesis and immunity because of the many similarities betweenRSV-induced disease in cotton rats and humans. Two important parallelsare the efficacy of neutralizing antibodies, and the enhanced lunghistopathology associated with formalin-inactivated RSV vaccination.Cotton rats are also more susceptible to RSV infection than other smallanimals such as mice.

To evaluate the immunogenicity of our RSV-F subunit vaccines, groups offemale cotton rats were vaccinated intramuscularly with various doses oftrimers (RSV-F-fusion-peptide-deletion-trunc) or rosettes(RSV-F-delp23-furdel-trunc, cleaved), each formulated with either alumor MF59. In all cases, a single immunization was sufficient to induceboth F-specific and neutralizing antibody in the serum when measuredthree weeks after the first vaccination (3wp1). All cotton rats weregiven a homologous booster immunization three weeks after the first, andthis resulted in a significant increase in F-specific IgG andneutralizing antibody when measured two weeks later (2wp2). Generally,the immunogenicity of rosettes was equal to or greater than that oftrimers, MF59 formulation enhanced titers more than alum formulation,and higher protein doses yielded higher titers, although there were someexceptions.

To determine the protective capacity of the subunit vaccines, all cottonrats were infected four weeks after the second vaccination with RSV bythe nasal route and the viral load in the lung was measured five dayslater by plaque assay. In all cases, subunit vaccination conferredprotection from challenge, as pulmonary viral loads in vaccinated cottonrats were greater than three orders of magnitude lower than unimmunized,but challenged control animals.

TABLE 4 F-specific serum IgG titers Protein F-specific serum IgGtiter^(a) Serum dose alum MF59 collected (μg) trimer rosette trimerrosette 3wp1 10 20276 36841 10251 22415 1 18341 20802 3712 28610 0.12698 6896 1065 8293 2wp2 10 103670 97174 130016 156144 1 142331 102405177441 299501 0.1 11581 34354 50238 111099 ^(a)geometric mean titer forindividual cotton rats (7-8 per group) trimer immunogen wasRSV-F-fusion-peptide-deletion-trunc rosette immonogen wasRSV-F-delp23-furdel-trun, cleaved.

TABLE 4A Lung viral titer 5 days post RSV challengea Protein dose viralvaccination (μg) titer^(b) none — 822760 trimer/alum 10 546 1 636 0.1903 rosette/alum 10 305 1 341 0.1 548 trimer/MF59 10 360 1 301 0.1 456rosette/MF59 10 244 1 257 0.1 716 aintranasal challenge with 1 × 10⁵plaque-forming units(pfu) of RSV Long ^(b)pfu/gram lung 5 days postchallenge Geometric mean titers of 7-8 individual cotton rats/group. Ifan individual animal had a titer of <203 (limit of detection) it wasassigned a titer of 100

TABLE 5 RSV serum neutralization titer Protein RSV serum neutralizationtiter^(a) Serum dose alum MF59 collected (μg) trimer rosette trimerrosette 3wp1 10 628 1050 578 229 1 208 633 165 205 0.1 57 200 51 65 2wp210 3669 4015 3983 3436 1 3369 2844 5728 3940 0.1 744 1902 2414 2093^(a)60% synctia reduction neutralization titers geometric mean titer fortwo pools of 3-4 cotton rats per group

Example 7—RSV RNA Vaccine

RNA Synthesis

Plasmid DNA encoding alphavirus replicon (FIGS. 4A-4F, SEQ ID NO:77)served as a template for synthesis of RNA in vitro. For theseexperiments the full length surface fusion glycoprotein of RSV (RSV-F)was used (FIGS. 4A-4F). Upon delivery of the replicons to eukaryoticcells, the positive-stranded RNA was translated to produce fournon-structural proteins, which together replicated the genomic RNA andtranscribed abundant subgenomic mRNAs encoding the heterologous geneproduct. Due to the lack of expression of the alphavirus structuralproteins, replicons are incapable of inducing the generation ofinfectious particles. A bacteriophage (T7 or SP6) promoter upstream ofthe alphavirus cDNA facilitates the synthesis of the replicon RNA invitro and the hepatitis delta virus (HDV) ribozyme immediatelydownstream of the poly(A)-tail generates the correct 3′-end through itsself-cleaving activity.

Following linearization of the plasmid DNA downstream of the HDVribozyme with a suitable restriction endonuclease, run-off transcriptswere synthesized in vitro using T7 or SP6 bacteriophage derivedDNA-dependent RNA polymerase. Transcriptions were performed for 2 hoursat 37° C. in the presence of 7.5 mM (T7 RNA polymerase) or 5 mM (SP6 RNApolymerase) of each of the nucleoside triphosphates (ATP, CTP, GTP andUTP) following the instructions provided by the manufacturer (Ambion,Austin, Tex.). Following transcription, the template DNA was digestedwith TURBO DNase (Ambion, Austin, Tex.). The replicon RNA wasprecipitated with LiCl and reconstituted in nuclease-free water.Uncapped RNA was capped post-transcripionally with Vaccinia CappingEnzyme (VCE) using the ScriptCap m⁷G Capping System (EpicentreBiotechnologies, Madison, Wis.) as outlined in the user manual.Post-transcriptionally capped RNA was precipitated with LiCl andreconstituted in nuclease-free water. The concentration of the RNAsamples was determined by measuring the optical density at 260 nm.Integrity of the in vitro transcripts was confirmed by denaturingagarose gel electrophoresis.

Lipid Nanoparticle (Liposome) Formulation RV01(01)

1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DlinDMA) was synthesizedusing a previously published procedure [Heyes, J., Palmer, L., Bremner,K., MacLachlan, I. Cationic lipid saturation influences intracellulardelivery of encapsulated nucleic acids. Journal of Controlled Release,107: 276-287 (2005)]. 1, 2-Diastearoyl-sn-glycero-3-phosphocholine(DSPC) was purchased from Genzyme. Cholesterol was obtained fromSigma-Aldrich (St. Lois, Mo.). 1, 2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000](ammonium salt) (PEG DMG 2000), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (ammonium salt) was obtained from Avanti Polar Lipids(Alabaster, Ala.).

Fresh lipid stock solutions in ethanol were prepared. 37 mg of DlinDMA,11.8 mg of DSPC, 27.8 mg of Cholesterol and 8.07 mg of PEG DMG 2000 wereweighed and dissolved in 7.55 mL of ethanol. The freshly prepared lipidstock solution was gently rocked at 37° C. for about 15 minutes to forma homogenous mixture. Then, 755 μL of the stock was added to 1.245 mLethanol to make a working lipid stock solution of 2 mL. This amount oflipid was used to form LNPs with 250 μg RNA at a 8:1 N:P (Nitrogen toPhosphate) ratio. The protonatable nitrogen on DlinDMA (the cationiclipid) and phosphates on the RNA are used for this calculation. Each μgof self-replicating RNA molecule was assumed to contain 3 nmoles ofanionic phosphate, each μg of DlinDMA was assumed to contain 1.6 nmolesof cationic nitrogen. A 2 mL working solution of RNA was also preparedfrom a stock solution of ˜1 μg/L in 100 mM citrate buffer (pH 6)(Teknova, Hollister, Calif.)). Three 20 mL glass vials (with stir bars)were rinsed with RNase Away solution (Molecular BioProducts, San Diego,Calif.) and washed with plenty of MilliQ water before use todecontaminate the vials of RNAses. One of the vials was used for the RNAworking solution and the others for collecting the lipid and RNA mixes(as described below). The working lipid and RNA solutions were heated at37° C. for 10 minutes before being loaded into 3cc luer-lok syringes (BDMedical, Franklin Lakes, N.J.). 2 mL of citrate buffer (pH 6) was loadedin another 3 cc syringe. Syringes containing RNA and the lipids wereconnected to a T mixer (PEEK™ 500 μm ID junction, Idex Health Science,Oak Harbor, Wash.) using FEP tubing([fluorinated ethylene-propylene] 2mm ID×3 mm OD, Idex Health Science, Oak Harbor, Wash.). The outlet fromthe T mixer was also FEP tubing (2 mm ID×3 mm). The third syringecontaining the citrate buffer was connected to a separate piece oftubing (2 mm ID×3 mm OD). All syringes were then driven at a flow rateof 7 mL/min using a syringe pump (kdScientific, model no. KDS-220,Holliston, Mass.). The tube outlets were positioned to collect themixtures in a 20 mL glass vial (while stirring). The stir bar was takenout and the ethanol/aqueous solution was allowed to equilibrate to roomtemperature for 1 hour. 4 ml of the mixture was loaded into a 5 ccsyringe (BD Medical), which was connected to a piece of FEP tubing (2 mmID×3 mm OD, Idex Health Science, Oak Harbor, Wash.) and in another 5 ccsyringe connected to an equal length of FEP tubing, an equal amount of100 mM citrate buffer (pH 6) was loaded. The two syringes were driven at7 mL/min flow rate using the syringe pump and the final mixturecollected in a 20 mL glass vial (while stirring). Next, the mixturecollected from the second mixing step (liposomes) were passed through aMustang Q membrane (an anion-exchange support that binds and removesanionic molecules, obtained from Pall Corporation, AnnArbor, Mich.,USA). Before passing the liposomes, 4 mL of 1 M NaOH, 4 mL of 1 M NaCland 10 mL of 100 mM citrate buffer (pH 6) were successively passedthrough the Mustang membrane. Liposomes were warmed for 10 minutes at37° C. before passing through the mustang filter. Next, liposomes wereconcentrated to 2 mL and dialyzed against 10-15 volumes of 1×PBS (fromTeknova) using the Tangential Flow Filtration (TFF) system beforerecovering the final product. The TFF system and hollow fiber filtrationmembranes were purchased from Spectrum Labs (Rancho Dominguez, Calif.)and were used according to the manufacturer's guidelines. Polysulfonehollow fiber filtration membranes (part number P/N: X1AB-100-20P) with a100 kD pore size cutoff and 8 cm² surface area were used. For in vitroand in vivo experiments, formulations were diluted to the required RNAconcentration with 1×PBS (from Teknova).

Method of Preparing Cationic Emulsion 17 (CNE17)

Squalene, sorbitan trioleate (Span 85), and polyoxy-ethylene sorbitanmonololeate (Tween 80) were obtained from Sigma (St. Louis, Mo., USA).1,2-Dioleoyl-3-trimethylammonium-propane (DOTAP) was purchased fromLipoid (Ludwigshafen Germany). Cationic nanoemulsions (CNEs) wereprepared similarly to charged MF59 as previously described with minormodifications Ott, et al. Journal of Controlled Release, 79(1-3):1-5(2002)). Briefly, oil soluble components (ie. Squalene, span 85,cationic lipids, lipid surfactants) were combined in a beaker, lipidcomponents were dissolved in chloroform (CHCl₃) or dichloromethane(DCM). The resulting lipid solution was added directly to the oil plusspan 85. The solvent was allowed to evaporate at room temperature for 2hours in a fume hood prior to combining the aqueous phase andhomogenizing the sample using an IKA T25 homogenizer at 24K RPM in orderto provide a homogeneous feedstock. The primary emulsions were passedthree to five times through a Microfluidezer M110S or M110PS homogenizerwith an ice bath cooling coil at a homogenization pressure ofapproximately 15k-20k PSI (Microfluidics, Newton, Mass.). The 20 mlbatch samples were removed from the unit and stored at 4° C. The tablebelow describes the composition of CNE17.

TABLE 6 Composition of CNE17 Cationic Lipid mg/ml + Squa- CNE (+) LipidSurfactant lene Buffer/water CNE17 DOTAP 1.40 0.5% SPAN 85 4.3% 10 mMcitrate (in DCM) 0.5% Tween 80 buffer pH 6.5

RNA Complexation

The number of nitrogens in solution were calculated from the cationiclipid concentration, DOTAP for example has 1 nitrogen that can beprotonated per molecule. The RNA concentration was used to calculate theamount of phosphate in solution using an estimate of 3 nmols ofphosphate per microgram of RNA. By varying the amount of RNA: Lipid theN/P ratio can be modified. RNA was complexed to CNE17 at anitrogen/phosphate ratios (N/P) of 10:1. Using these values the RNA wasdiluted to the appropriate concentration in RNase free water and addeddirectly into an equal volume of emulsion while vortexing lightly. Thesolution was allowed to sit at room temperature for approximately 2hours. Once complexed the resulting solution was diluted to the requiredconcentration prior to administration.

Electroporation

Electroporation was a very effective method for the delivery of pDNAvaccines and this technique was used to deliver self-replicating RNA.Mice were anesthetized under isofluorane, both hind legs were closelyshaven to expose the area on the limb to be treated. A dose of 30 ul ofvaccine was injected to the calf muscle of the hind limb using a ½ccinsulin syringe. The muscle was electroporated using the Elgen® DNADelivery System (Inovio, San Diego). The instrument parameters are asfollows: 60V, 2 pulses each at 60 ms. Another dose was similarlydelivered to the second limb, followed by electroporation.

Viral Replicon Particles (VRP)

To compare RNA vaccines to traditional RNA-vectored approaches forachieving in vivo expression of reporter genes or antigens, we utilizedviral replicon particles (VRPs) produced in BHK cells by the methodsdescribed by Perri et al. In this system, the antigen (or reporter gene)replicons consisted of alphavirus chimeric replicons (VCR) derived fromthe genome of Venezuelan equine encephalitis virus (VEEV) engineered tocontain the 3′ terminal sequences (3′ UTR) of Sindbis virus and aSindbis virus packaging signal (PS) (see FIG. 2 of Perri et al). Thesereplicons were packaged into VRPs by co-electroporating them into babyhamster kidney (BHK) cells along with defective helper RNAs encoding theSindbis virus capsid and glycoprotein genes (see FIG. 2 of Perri et al).The VRPs were then harvested and titrated by standard methods andinoculated into animals in culture fluid or other isotonic buffers.Perri S, Greer C E, Thudium K, Doe B, Legg H, Liu H, Romero R E, Tang Z,Bin Q, Dubensky T W, Jr. et al (2003) An alphavirus replicon particlechimera derived from venezuelan equine encephalitis and sindbis virusesis a potent gene-based vaccine delivery vector. J Virol 77: 10394-10403

RSV F Trimer Subunit Vaccine

The RSV F trimer is a recombinant protein comprising the ectodomain ofRSV F with a deletion of the fusion peptide region preventingassociation with other trimers. The resulting construct forms ahomogeneous trimer, as observed by size exclusion chromatography, andhas an expected phenotype consistent with a postfusion F conformation asobserved by electron microscopy. The protein was expressed in insectcells and purified by virtue of a HIS-tagged in fusion with theconstruct's C-terminus followed by size exclusion chromatography usingconventional techniques. The resulting protein sample exhibits greaterthan 95% purity. For the in vivo evaluation of the F-subunit vaccine,100 μg/mL trimer protein was adsorbed on 2 mg/mL alum using 10 mMHistidine buffer, pH 6.3 and isotonicity adjusted with sodium chlorideto 150 mM. F-subunit protein was adsorbed on alum overnight with gentlestirring at 2-8° C. The pH and osmolality of the final vaccine wastargeted as 6.5-7.5 and 240-360 mOsm/kg. The vaccine was characterizedfor protein adsorption by SDS-PAGE (Invitrogen Corporation, USA) and forendotoxin content by LAL assay (Charles River Laboratories, USA). Thevaccine was mixed by gentle inversion prior to immunization.

Murine Immunogenicity Studies

Groups of 10 female BALB/c mice aged 8-10 weeks and weighing about 20grams were immunized at day 0 and day 21 with bleeds taken at days 14,35 and 49. All animals were injected in the quadriceps in the two hindlegs each getting an equivalent volume (50 μl per site) for a total of100 μl of vaccine to deliver 10 μg antigen dose. When measurement of Tcell responses was required, spleens were harvested at day 35 or 49.

Vaccination and Challenge of Cotton Rats

Female cotton rats (Sigmodon hispidis) were obtained from HarlanLaboratories. All experiments were approved and performed according toNovartis Animal Care and Use Committee. Groups of animals were immunizedintramuscularly (i.m., 100 μl) with the indicated vaccines on days 0 and21. Serum samples were collected 2 weeks after each immunization.Immunized or unvaccinated control animals were challenged intranasally(i.n.) with 1×10⁵ PFU RSV 4 weeks after the final immunization. Bloodcollection and RSV challenge were performed under anesthesia with 3%isoflurane using a precision vaporizer.

Mouse T Cell Function Assays

Intracellular Cytokines Immunofluorescence Assay

Two to five spleens from identically vaccinated BALB/c mice were pooledand single cell suspensions were prepared for culture. Twoantigen-stimulated cultures and two unstimulated cultures wereestablished for each splenocyte pool. Antigen-stimulated culturescontained 1×10⁶ splenocytes, RSV F peptide 85-93 (1×10⁻⁶ M), RSV Fpeptide 249-258 (1×10⁻⁶ M), RSV F peptide 51-66 (1×10⁻⁶ M), anti-CD28mAb (1 mcg/mL), and Brefeldin A (1:1000). Unstimulated cultures did notcontain RSV F peptides, and were otherwise identical to the stimulatedcultures. After culturing for 6 hours at 37° C., cultures were processedfor immunofluorescence. Cells were washed and then stained withfluorecently labeled anti-CD4 and anti-CD8 monoclonal antibodies (mAb).Cells were washed again and then fixed with Cytofix/cytoperm for 20minutes. The fixed cells were then washed with Perm-wash buffer and thenstained with fluorescently labeled mAbs specific for IFN-g, TNF-a, IL-2,and IL-5. Stained cells were washed and then analyzed on an LSR II flowcytometer. FlowJo software was used to analyze the acquired data. TheCD4+8− and CD8+4− T cell subsets were analyzed separately. For eachsubset in a given sample the % cytokine-positive cells was determined.The % RSV F antigen-specific T cells was calculated as the differencebetween the % cytokine-positive cells in the antigen-stimulated culturesand the % cytokine-positive cells in the unstimulated cultures. The 95%confidence limits for the % antigen-specific cells were determined usingstandard methods (Statistical Methods, 7^(th) Edition, G. W. Snedecorand W. G. Cochran).

Secreted cytokines assay

The cultures for the secreted cytokines assay were similar to those forthe intracellular cytokines immunofluorescence assay except thatBrefeldin A was omitted. Culture supernatants were collected afterovernight culture at 37° C., and were analyzed for multiple cytokinesusing mouse Th1/Th2 cytokine kits from Meso Scale Discovery. The amountof each cytokine per culture was determined from standard curvesproduced using purified, recombinant cytokines supplied by themanufacturer.

RSV F-Specific ELISA

Individual serum samples were assayed for the presence of RSV F-specificIgG by enzyme-linked immunosorbent assay (ELISA). ELISA plates (MaxiSorp96-well, Nunc) were coated overnight at 4° C. with 1 μg/ml purified RSVF (delp23-furdel-trunc uncleaved) in PBS. After washing (PBS with 0.1%Tween-20), plates were blocked with Superblock Blocking Buffer in PBS(Thermo Scientific) for at least 1.5 hours at 37° C. The plates werethen washed, serial dilutions of serum in assay diluent (PBS with 0.1%Tween-20 and 5% goat serum) from experimental or control cotton ratswere added, and plates were incubated for 2 hours at 37° C. Afterwashing, plates were incubated with horse radish peroxidase(HRP)-conjugated chicken anti-cotton rat IgG (Immunology ConsultantsLaboratory, Inc, diluted 1:5,000 in assay diluent) for 1 hour at 37° C.Finally, plates were washed and 100 μl of TMB peroxidase substratesolution (Kirkegaard & Perry Laboratories, Inc) was added to each well.Reactions were stopped by addition of 100 μl of 1M H₃PO₄, and absorbancewas read at 450 nm using a plate reader. For each serum sample, a plotof optical density (OD) versus logarithm of the reciprocal serumdilution was generated by nonlinear regression (GraphPad Prism). Titerswere defined as the reciprocal serum dilution at an OD of approximately0.5 (normalized to a standard, pooled sera from RSV-infected cotton ratswith a defined titer of 1:2500, that was included on every plate).

Micro Neutralization Assay

Serum samples were tested for the presence of neutralizing antibodies bya plaque reduction neutralization test (PRNT). Two-fold serial dilutionsof HI-serum (in PBS with 5% HI-FBS) were added to an equal volume of RSVLong previously titered to give approximately 115 PFU/25 μl. Serum/virusmixtures were incubated for 2 hours at 37° C. and 5% CO₂, to allow virusneutralization to occur, and then 25 μl of this mixture (containingapproximately 115 PFU) was inoculated on duplicate wells of HEp-2 cellsin 96 well plates. After 2 hours at 37° C. and 5% CO₂, the cells wereoverlayed with 0.75% Methyl Cellulose/EMEM 5% HI-FBS and incubated for42 hours. The number of infectious virus particles was determined bydetection of syncytia formation by immunostaining followed by automatedcounting. The neutralization titer is defined as the reciprocal of theserum dilution producing at least a 60% reduction in number of synctiaper well, relative to controls (no serum).

Viral Load

Viral load in the lung was determined by plaque assay. Specifically,lungs were harvested 5 days post RSV infection and one right lobe wasplaced into 2.5 ml Dulbecco's Modified Eagle Medium (DMEM, Invitrogen)with 25% sucrose and disrupted with a tissue homogenizer. Cell-freesupernatants from these samples were stored at −80° C. To assay forinfectious virus, dilutions of clarified lung homogenate (in PBS with 5%heat-inactivated fetal bovine serum, HI-FBS) were inoculated onconfluent HEp-2 cell monolayers in a volume of 200 μl/well of a 12-wellplate. After 2 hours with periodic gentle rocking (37° C., 5% CO₂), theinoculum was removed, and cells were overlaid with 1.5 ml of 1.25%SeaPlaque agarose (Lonza) in Eagle's Minimal Essential Medium (EMEM,Lonza) supplemented with 5% HI-FBS, glutamine, and antibiotics. After3-4 days of incubation, cells were again overlaid with 1 ml of 1.25%agarose in EMEM (Sigma) containing 0.1% neutral red (Sigma). Plaqueswere counted one day later with the aid of a light box.

Cotton Rat Lung Pathology

Five days after RSV challenge lungs were harvested and 4 lobes from eachanimal were collected and fixed with 10% neutral buffered formalin (NBF)by gentle intratracheal instillation followed by immersion fixation.Tissues were processed routinely to prepare hematoxylin & eosin-stainedsections for microscopic examination. Findings were evaluated using amodification of previously published criteria [Prince G A, et al., 2001]for the following parameters: peribronchiolitis, alveolitis, bronchitis,perivascular cellular infiltrates, and interstitial pneumonitis. Lesionswere graded on a 4-point semiquantitative scale. Minimal (+) changecontained one or a few small foci; mild (++) change was composed ofsmall- to medium-size foci; moderate (+++) change contained frequentand/or moderately-sized foci; and marked (++++) change showed extensiveto confluent foci affecting most/all of the tissue.

Example 7

A—Cotton Rat RSV Challenge Study (CRIS14)

The A317 replicon, which expresses the surface fusion glycoprotein ofRSV (RSV-F) was used for this experiment. Cotton rats (Sigmodonhispidus), 8 animals per group, were given bilateral intramuscularvaccinations (50 μL per leg) on days 0 and 21 with nakedself-replicating RNA (A317, 1 μg or 10 μg), self-replicating RNAformulated in LNP [RV01(01), A317, 0.1 μg or 1 μg), VRPs (5×10⁶ IU)expressing RSV-F, F-trimer/alum subunit (10 μg), or formalin inactivatedRSV vaccine (5200 FI-pfu). Serum was collected for antibody analysis ondays 14 (2wp1) and 35 (2wp2). All animals were challenged with 1×10⁵ pfuRSV intranasally on day 49 and lungs were collected on day 54 (5dpc) fordetermination of viral load and lung pathology.

Results

TABLE 7 F-specific serum IgG titers on day 14 and 35 F-specific IgGF-specific IgG vaccine dose 2wp1 2wp2 A317 10 μg 198 1599 A317 1 μg 78526 CNE17 1 μg 408 4918 CNE17 0.1 μg 325 2512 RV01(01) 1 μg 531 4351RV01(01) 0.1 μg 134 1033 VRP 5 × 106 IU 961 5864 F-trimer/alum 10 μg3526 111893 FI-RSV 5200 FI-pfu 17 2074 none 5 5Table 7. F-specific serum IgG titers of cotton rats (Sigmodon hispidus),8 animals per group, after intramuscular vaccinations on days 0 and 21.Serum was collected for antibody analysis on days 14 (2wp1) and 35(2wp2), all animals were challenged with 1×10⁵ pfu RSV intranasally onday 49. Lungs were collected on day 54 (5dpc) for determination of viralload and lung pathology. Data are represented as geometric mean titersof 8 individual cotton rats per group. If an individual animal had atiter of <25 (limit of detection) it was assigned a titer of 5.

TABLE 8 RSV serum neutralization titers on days 14 and 35 PRNT60 PRNT60vaccine dose 2wp1 2wp2 A317 10 μg 78 240 A317 1 μg 58 70 CNE17 1 μg 91269 CNE17 0.1 μg 63 145 RV01(01) 1 μg 103 667 RV01(01) 0.1 μg 46 130 VRP5 × 10⁶ IU 149 683 F-trimer/alum 10 μg 142 >5120 FI-RSV 5200 FI-pfu 2838 none 30 <20Table 8. RSV serum neutralization titers of cotton rats (Sigmodonhispidus), 8 animals per group, after intramuscular vaccinations on days0 and 21. Serum was collected for analysis on days 14 (2wp1) and 35(2wp2). Data are represented as 60% plaque reduction neutralizationtiters. Geometric mean titer of 2 pools of 4 cotton rats per group. Ifan individual animal had a titer of <25 (limit of detection) it wasassigned a titer of 5.

TABLE 9 Lung viral titers 5 days post RSV challenge pfu/g lung vaccinedose 5dpc A317 10 μg 397 A317 1 μg 659 CNE17 1 μg 414 CNE17 0.1 μg 572RV01(01) 1 μg 445 RV01(01) 0.1 μg 716 VRP 5 × 10⁶ IU 359 F-trimer/alum10 μg 190 FI-RSV 5200 FI-pfu 5248 none (challenged) 728618Table 9: Lung viral titers 5 days post RSV challenge of cotton rats(Sigmodon hispidus), 8 animals per group, after intramuscularvaccinations on days 0 and 21. All animals were challenged with 1×10⁵pfu RSV intranasally on day 49. Lungs were collected on day 54 (5dpc)for determination of viral load and lung pathology. Data are representedas plaque forming units per gram lung as determined by plaque assay.Geometric mean titers of 8 individual cotton rats per group. In anindividual animal had a titer of <200 (limit of detection) it wasassigned a titer of 100.

TABLE 10 Lung alveolitis scores 5 days post RSV challenge # of cottonrats with indicated alveolitis score vaccine dose 0 1 2 3 4 A317l 10 μg8 A317l 1 μg 8 CNE17 1 μg 8 CNE17 0.1 μg 7 1 RV01(01) 1 μg 6 2 RV01(01)0.1 μg 8 VRP 5 × 10⁶ IU 3 4 1 F-trimer/alum 10 μg 7 1 FI-RSV 5200 FI-pfu1 4 3 none (challenged) 5 3Table 10. Lung alveolitis 5 days post RSV challenge of cotton rats(Sigmodon hispidus), 8 animals per group, after intramuscularvaccinations on days 0 and 21. All animals were challenged with 1×10⁵pfu RSV intranasally on day 49. Lungs were collected on day 54 (5dpc)for determination of viral load and lung pathology. Lesions were gradedon a 4-point semiquantitative scale. Minimal (1) change contained one ora few small foci; mild (2) change was composed of small- to medium-sizefoci; moderate (3) change contained frequent and/or moderately-sizedfoci; and marked (4) change showed extensive to confluent foci affectingmost/all of the tissue.

Conclusions

One objective of this study was to determine the immunogenicity andprotective capacity of replicon RNA in the cotton rat RSV model. Anotherobjective was to evaluate the effect of Liposomes and CNE17 formulationson vaccine immunogenicity and efficacy. Unformulated replicon RNAinduced serum F-specific IgG and RSV neutralizing antibodies after onevaccination, and this response was boosted by a second vaccination.Liposomes and CNE17 formulations were similarly effective in this model,boosting F-specific IgG titers to 1 μg replicon RNA approximately 8-foldand neutralization titers by 4-10-fold (CNE17 and Liposomes,respectively) after the second vaccination. All replicon RNA vaccinesprovided protection from a nasal RSV challenge, reducing the lung viralload great than 3 order of magnitude when measured 5 days later. Themagnitude and protective capacity of the immune response generated by 1μg replicon RNA formulated with Liposomes was within 2-fold the responseelicited by 5×10⁶ VRPs. The alum adjuvanted trimer subunit elicited thehighest total anti-F IgG ELISA titers, elicited the highestneutralization titers, and elicited the greatest degree of protectionfrom RSV titers in the lung on challenge of any of the vaccinepreparations tested in this study.

Example 7B—RSV-F Immunogenicity Study (10-1001)

The A317 replicon that expresses the surface fusion glycoprotein of RSV(RSV-F) was used for this experiment. BALB/c mice, 10 animals per group,were given bilateral intramuscular vaccinations (50 μL per leg) on days0 and 21 with VRP's expressing RSV-F (1×10⁶ IU), naked self-replicatingRNA (A317, 1 μg), self-replicating RNA delivered using electroporation(A317, 10 μg), self-replicating RNA formulated in liposomes [RV01(01),A317, 0.1 μg or 1 μg) and self-replicating RNA formulated with CNE17(A317, 0.1 μg or 1 μg). Serum was collected for antibody analysis ondays 14 (2wp1), 35 (2wp2) and 49 (4wp2). Spleens were harvested from 5mice per group at day 49 (4wp2) for T cell analysis.

Results

TABLE 11 F-specific serum IgG titers on day 14 1 μg A317 0.1 μg RV01(01)1 μg RV01(01) 0.1 μg CNE17 1 μg CNE17 10 μg A317 + EP 1E6 IU VRP 52914385 19299 2429 3373 5 6041 1530 10713 19170 2060 4417 88 4912 273412756 13860 2012 1927 964 12923 2503 11546 17352 1887 3597 7235 70755539 15300 22094 3174 5731 2558 6829 1033 14072 21213 3904 2852 51054885 5110 18274 17915 1481 3739 9806 3680 1106 7873 15659 2345 4904 27879813 1493 17175 6669 3084 3824 2576 8631 3456 19730 13259 2497 3004 18586314 GMT 1980 13731 15903 2398 3590 1180 6685Table 11. (10-1001) F-specific serum IgG titers of mice, 10 animals pergroup, 14 days after intramuscular vaccination. Data are represented astiters for individual mice and the geometric mean titers of 10individual mice per group. If an individual animal had a titer of <25(limit of detection) it was assigned a titer of 5.

TABLE 12 F-specific serum IgG titers on day 35 1 μg A317 0.1 μg RV01(01)1 μg RV01(01) 0.1 μg CNE17 1 μg CNE17 10 μg A317 + EP 1E6 IU VRP 958128208 227021 48079 8473 14612 813045 12518 191729 212911 17589 5855622805 365485 4839 315786 303987 8522 12053 32156 961601 10128 218147335071 10985 20395 24090 349215 18451 225622 155893 30801 51514 31053297526 9805 182693 519162 13372 26348 18105 207652 19154 185342 1693325137 80686 23918 1580066 4490 82744 489441 47173 21014 9091 900889 14674190010 131361 78232 61076 21006 822285 15223 553164 254500 24135 254999835 587121 GMT 8532 201892 253687 20767 29111 19117 579033Table 12. (10-1001) F-specific serum IgG titers of mice, 10 animals pergroup, after intramuscular vaccinations on days 0 and 21. Serum wascollected for antibody analysis on day 35 (2wp2). Data are representedas titers for individual mice and the geometric mean titers of 10individual mice per group. If an individual animal had a titer of <25(limit of detection) it was assigned a titer of 5.

TABLE 13 F-specific serum IgG titers on day 49 1 μg A317 0.1 μg RV01(01)1 μg RV01(01) 0.1 μg CNE17 1 μg CNE17 10 μg A317 + EP 1E6 IU VRP 1248140407 133787 52747 34245 30388 366771 12441 155669 182995 29352 12803020768 209400 4967 203059 211020 10857 17016 53763 360615 14536 134253488698 28800 57250 28373 191475 31556 370726 158816 44613 76576 34318139148 13815 184738 185184 20314 42357 35736 63839 20495 141644 1030264546 101445 34611 192101 4800 76711 312096 27476 47285 10138 17785819159 143275 139811 68386 55865 23958 130218 26836 479594 230331 2436052871 13624 174378 GMT 10947 177168 194350 24891 53615 25888 180420Table 13. (10-1001) F-specific serum IgG titers of mice, 10 animals pergroup, after intramuscular vaccinations on days 0 and 21. Serum wascollected for antibody analysis on days 49 (4wp2). Data are representedas titers for individual mice and the geometric mean titers of 10individual mice per group. If an individual animal had a titer of <25(limit of detection) it was assigned a titer of 5.

TABLE 14 RSV serum neutralization titers on day 35 A317, 1 μg RV01(01)0.1 μg RV01(01) 1 μg CNE17 0.1 μg CNE17 1 μg VRP 1E6 IU 2wp2 2wp2 2wp22wp2 2wp2 2wp2 NA 143 106 NA NA 265 NA 272 62 NA NA 73 NA 294 <40 NA NA77 NA 814 334 NA NA 140 NA 67 86 NA NA 290 NA 420 125 NA NA 134 NA <40566 NA NA 466 NA 104 292 NA NA 127 NA 241 <40 NA NA 75 NA 223 44 NA NA77 GMT NA 176 96 NA NA 139Table 14: (10-1001) RSV serum neutralization titers of mice, 10 animalsper group, after intramuscular vaccinations on days 0 and 21. Serum wascollected for analysis on day 35 (2wp2). Data are represented as 60%plaque reduction neutralization titers of individual mice and thegeometric mean titer of 10 individual mice per group. If an individualanimal had a titer of <40 (limit of detection) it was assigned a titerof 20. NA=not assayed.

TABLE 15 RSV serum neutralization titers on day 49 A317, 1 μg RV01(01)0.1 μg RV01(01) 1 μg CNE17 0.1 μg CNE17 1 μg VRP 1E6 IU 4wp2 4wp2 4wp24wp2 4wp2 4wp2 <40 194 82 <40 <40 161 <40 272 165 <40 70 64 <40 142 <40<40 <40 126 <40 881 442 <40 76 151 <40 61 108 42 57 194 <40 426 156 52<40 123 <40 78 814 <40 <40 1033 <40 <40 291 173 <40 174 <40 246 103 <40<40 122 <40 574 396 <40 <40 76 GMT <40 231 215 29 29 155Table 15: (10-1001) RSV serum neutralization titers of mice, 10 animalsper group, after intramuscular vaccinations on days 0 and 21. Serum wascollected for analysis on day 49 (4wp2). Data are represented as 6000plaque reduction neutralization titers of individual mice and thegeometric mean titer of 10 individual mice per group. If an individualanimal had a titer of <40 (limit of detection) it was assigned a titerof 20. NA=not assayed.

TABLE 16 T cell responses at day 49 4wp2 splenic CD4+CD8− splenic Tcells: % cytokine-positive CD4 T cell and specific for RSV F51-66peptide responses IFNg+ IL2+ IL5+ TNFa+ VRP 1E6 IU 0.07 ± 0.06 0.04 ±0.05 0.00 ± 0.02 0.10 ± 0.04 1 μg A317 0.00 ± 0.05 0.05 ± 0.04 0.00 ±0.01 0.03 ± 0.02 RV01(01) 0.04 ± 0.06 0.07 ± 0.05 0.00 ± 0.01 0.09 ±0.03 1 μg RV01(01) 0.06 ± 0.05 0.08 ± 0.04 0.00 ± 0.01 0.10 ± 0.03 0.1μg CNE17 0.00 ± 0.05 0.04 ± 0.04 0.00 ± 0.01 0.05 ± 0.02 1 μg CNE17 0.00± 0.05 0.02 ± 0.04 0.00 ± 0.01 0.02 ± 0.02 0.1 μg 10 μg 0.02 ± 0.06 0.04± 0.04 0.01 ± 0.01 0.05 ± 0.03 vA317 + EP none 0.04 ± 0.06 0.00 ± 0.050.00 ± 0.02 0.00 ± 0.01Table 16. (10-1001) Frequencies of RSV F-specific CD4+ splenic T cellson day 49 (4wp2). Shown are net (antigen-specific) cytokine-positivefrequency (%)±95% confidence half-interval. Net frequencies shown inbold indicate stimulated responses that were statistically significantly>0.

TABLE 17 T cell responses at day 49 4wp2 splenic CD8+CD4− splenic Tcells: % cytokine-positive CD8 T cell and specific for RSV F peptidesF85-93 and F249-258 responses IFNg+ IL2+ IL5+ TNFa+ VRP 1E6 IU 3.48 ±0.29 1.21 ± 0.18 −0.03 ± 0.05 3.31 ± 0.28 1 μg A317 0.74 ± 0.15 0.46 ±0.11 −0.03 ± 0.04 0.70 ± 0.14 RV01(01) 3.69 ± 0.28 1.43 ± 0.18 −0.01 ±0.04 3.44 ± 0.27 1 μg RV01(01) 2.52 ± 0.23 1.10 ± 0.15  0.03 ± 0.03 2.31± 0.22 0.1 μg CNE17 1.25 ± 0.17 0.60 ± 0.12  0.01 ± 0.03 1.15 ± 0.16 1μg CNE17 0.89 ± 0.15 0.49 ± 0.11 −0.03 ± 0.04 0.83 ± 0.14 0.1 μg 10 μg0.85 ± 0.15 0.53 ± 0.11  0.01 ± 0.04 0.72 ± 0.15 A317 + EP none 0.01 ±0.07 0.00 ± 0.05 −0.02 ± 0.05 0.02 ± 0.06Table 17. (10-1001) Frequencies of RSV F-specific CD8+ splenic T cellson day 49 (4wp2). Shown are net (antigen-specific) cytokine-positivefrequency (%)±95% confidence half-interval. Net frequencies shown inbold indicate stimulated responses that were statistically significantly>0.

Conclusions

Liposome formulation significantly enhanced immunogenicity, asdetermined by increased F-specific IgG titers (8-30-fold increase),neutralization titers, and CD4 and CD8 T cell responses, relative to thenaked RNA control. Surprisingly, the F-specific IgG titers andneutralization titers for RV01(01) at both the 0.1 and 1.0 μg doses wereequivalent to the VRP (1×10⁶ IU). T cell responses for the LNPformulation were equivalent at the higher dose to the VRP (1×10⁶ IU).Formulation of the self-replicating RNA with CNE17 significantlyenhanced immunogenicity, as determined by increased F-specific IgGtiters (2-5-fold increase), neutralization titers, and CD4 and CD8 Tcell responses, relative to the naked RNA control. Electroporation ofRNA enhanced immunogenicity relative to the naked RNA control, but wassignificantly lower than Liposome delivery.

Example 7C—RSV-F Immunogenicity Study (10-1018)

The A317 replicon that expresses the surface fusion glycoprotein of RSV(RSV-F) was used for this experiment. BALB/c mice, 8 animals per group,were given bilateral intramuscular vaccinations (50 μL per leg) on days0 and 21 with VRP's expressing RSV-F (1×10⁶ IU), naked self-replicatingRNA (A306, 1, 0.1, 0.01 μg) and self-replicating RNA formulated inliposomes (RV01(01) using method 1 (A317, 10.0, 1.0, 0.1, 0.01 μg).Serum was collected for antibody analysis on days 14 (2wp1) and (2wp2).Spleens were harvested from 5 mice per group at day 49 (4wp2) for T cellanalysis.

Results

RV01(01) liposome formulation had a Z average particle diameter of 158nm with a polydispersity index of 0.14, the encapsulation efficiency was96%. F-specific serum IgG titers on day 14 and 35 are shown in tables 18and 19 and T cell responses at day 49 are shown in tables 20 and 21.

TABLE 18 F-specific serum IgG titers on day 14 and 35 A317 VRP 1E6 IU 1μg 0.1 μg 0.01 μg 2wp1 2wp2 2wp1 2wp2 2wp1 2wp2 2wp1 2wp2 6334 39539 7724687 5 2334 143 1377 1500 14895 5 142 5 161 5 333 5450 38252 109 2972 5145 5 5 1835 12831 5 3674 5 97 5 5 2277 30326 5 5003 5 1077 5 175 281833437 663 8258 221 457 5 5 2405 22551 257 845 5 1558 5 456 2137 19179 51765 5 5 5 180 GMT 2735 24427 41 2144 8 259 8 73Table 18: (10-1018) F-specific serum IgG titers of mice, 8 animals pergroup, after intramuscular vaccinations on days 0 and 21. Serum wascollected for antibody analysis on days 14 (2wp1) and 35 (2wp2). Dataare represented as individual mice and the geometric mean titers of 8individual cotton rats per group. If an individual animal had a titer of<25 (limit of detection) it was assigned a titer of 5.

TABLE 19 F-specific serum IgG titers on day 14 and 35 RV01(01) 10 μg 1μg 0.1 μg 0.01 μg 2wp1 2wp2 2wp1 2wp2 2wp1 2wp2 2wp1 2wp2 5880 826897255 45018 4072 22174 619 2872 6126 42529 3009 22288 3974 27730 474 36038069 76263 5385 23561 3272 15328 914 2692 5966 108234 4148 53675 396824456 2011 11542 8590 57912 4210 37004 4950 13014 903 4684 7172 741622179 24179 2856 13694 1575 6720 8072 122796 1640 5994 4073 17849 43816514 8706 83601 5725 28760 3797 17342 1058 13665 GMT 7235 77338 378325790 3826 18325 879 6235Table 19: Continued from 23A. (10-1018) F-specific serum IgG titers ofmice, 8 animals per group, after intramuscular vaccinations on days 0and 21. Serum was collected for antibody analysis on days 14 (2wp1) and35 (2wp2). Data are represented as individual animals and the geometricmean titers (GMT) of 8 individual cotton rats per group. If anindividual animal had a titer of <25 (limit of detection) it wasassigned a titer of 5.

TABLE 20 T cell responses at day 49 4wp2 splenic CD4+CD8− splenic Tcells: % cytokine-positive T cell and specific for RSV F51-66 peptideresponses IFNg+ IL2+ IL5+ TNFa+ VRP 1E6 IU 0.00 ± 0.02 0.07 ± 0.02 0.00± 0.01 0.07 ± 0.03 1 μg A317 0.01 ± 0.01 0.03 ± 0.02 0.00 ± 0.01 0.03 ±0.02 0.1 μg A317 0.00 ± 0.01 0.01 ± 0.01 0.00 ± 0.00 0.01 ± 0.01 0.01 μgA317 0.00 ± 0.00 0.01 ± 0.01 0.01 ± 0.01 0.00 ± 0.01 RV01(01), 0.02 ±0.01 0.05 ± 0.02 0.00 ± 0.00 0.06 ± 0.02 10 μg RV01(01), 0.03 ± 0.020.08 ± 0.02 0.00 ± 0.01 0.09 ± 0.02 1 μg RV01(01), 0.02 ± 0.01 0.03 ±0.01 0.00 ± 0.01 0.03 ± 0.02 0.1 μg RV01(01), 0.00 ± 0.00 0.02 ± 0.020.01 ± 0.01 0.02 ± 0.02 0.01 μg none 0.00 ± 0.00 0.00 ± 0.01 0.00 ± 0.010.01 ± 0.01Table 20. Frequencies of RSV F-specific CD4+ splenic T cells on day 49(Expt. 10-1018, 4wp2). Shown are net (antigen-specific)cytokine-positive frequency (%)±95% confidence half-interval. Netfrequencies shown in bold indicate stimulated responses that werestatistically significantly >0.

TABLE 21 T cell responses at day 49 4wp2 splenic CD8+CD4− splenic Tcells: % cytokine-positive T cell and specific for RSV F peptides F85-93and F249-258 responses IFNg+ IL2+ IL5+ TNFa+ VRP 1E6 IU 2.45 ± 0.21 0.58± 0.10 0.00 ± 0.01 2.64 ± 0.21 1 μg A317 1.68 ± 0.17 0.45 ± 0.09 0.00 ±0.02 1.75 ± 0.18 0.1 μg A317 0.21 ± 0.07 0.08 ± 0.04 0.01 ± 0.02 0.30 ±0.08 0.01 μg A317 0.06 ± 0.05 0.05 ± 0.03 0.01 ± 0.02 0.16 ± 0.06RV01(01), 3.32 ± 0.23 0.69 ± 0.11 0.00 ± 0.02 3.90 ± 0.25 10 μgRV01(01), 1.81 ± 0.17 0.59 ± 0.10 0.00 ± 0.02 2.04 ± 0.20 1 μg RV01(01),0.91 ± 0.12 0.32 ± 0.07 0.00 ± 0.01 1.06 ± 0.14 0.1 μg RV01(01), 0.58 ±0.10 0.33 ± 0.08 0.00 ± 0.01 0.64 ± 0.11 0.01 μg none 0.01 ± 0.02 0.01 ±0.01 0.00 ± 0.01 0.00 ± 0.05Table 21. F-specific splenic CD8⁺ T cell frequencies on day 49 (Expt.10-1018, 4wp2). Shown are net (antigen-specific) cytokine-positivefrequency (%)±95% confidence half-interval. Net frequencies shown inbold indicate stimulated responses that were statistically significantly>0.

Conclusions

Liposome formulation significantly enhanced immunogenicity, asdetermined by increased F-specific IgG titers and T cell frequencies,relative to the naked RNA controls. The F-specific IgG titers and CD8 Tcell frequencies for RV01(01) at the 10 μg RNA dose were enhancedrelative to the VRP group (1×10⁶ IU).

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The entire teachings of all documents cited herein are herebyincorporated herein by reference.

1.-6. (canceled)
 7. An RSV F polypeptide which; has the transmembraneregion and cytoplasmic tail deleted; wherein the RSV F polypeptide issoluble, contains an added oligomerization sequence, and has one or moreadditional mutation(s) which stabilizes the RSV F polypeptide in itspre-fusion conformation; wherein the one or more additional mutation(s)are 2, 3, 4 or 5, single amino acid substitutions relative to a naturalRSV sequence; wherein the RSV F polypeptide comprises a foldontrimerization sequence; wherein the RSV F polypeptide comprises amutation in the HRB domain wherein the mutation in the HRB domain isbetween residues 474 and 525; wherein the mutation in the HRB domaindisfavours the post-fusion conformation of the RSV F polypeptide.
 8. TheRSV F polypeptide of claim 7, which further comprises cysteine bridgesat the surface or within the core.
 9. The RSV F polypeptide of claim 7,which further comprises a mutation in the HRA domain, wherein themutation disfavours the post-fusion conformation of the RSV Fpolypeptide.
 10. The RSV F polypeptide of claim 7, which furthercomprises a mutation between residues 154 and 206, wherein the mutationdisfavours the post-fusion conformation of the RSV F polypeptide.
 11. Anucleic acid encoding the RSV F polypeptide of claim
 7. 12. A vectorcomprising the nucleic acid of claim
 11. 13. An immunogenic compositioncomprising the RSV F polypeptide of claim
 7. 14. An immunogeniccomposition comprising the nucleic acid of claim
 11. 15. An immunogeniccomposition comprising the vector of claim
 12. 16. A method ofimmunizing a patient against RSV infection, which comprisesadministering an effective amount of the immunogenic composition ofclaim 13 to said patient.
 17. The RSV F polypeptide of claim 7, whereinthe one or more additional mutation(s) are 4 or 5 single amino acidsubstitutions.
 18. The RSV F polypeptide of claim 7, wherein themutation in the HRB domain is between residues 484 and 517.